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Tumor modeling in mice with conditional retinoblastoma gene deficiency

Vooijs, M.A.G.G.

Link to publication

Citation for published version (APA):Vooijs, M. A. G. G. (2001). Tumor modeling in mice with conditional retinoblastoma gene deficiency.

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Download date: 27 Jan 2021

Chapterr 4

Tumorigenesiss in mice after somatic deletion of Rb inn Interphotoreceptor Retinol Binding Protein-expressing cells.

M.. Vooijs, te Riele H., van der Valk M. and Bems A.

SubmittedSubmitted for publication

47 7

Tumorigenesi ss in mic e after somati c deletio n of Rb in Interphoto -recepto rr Retino l Bindin g Protein-expressin g cell s

Mar cc Vooijs1, Hein te Riele2, Marti n van derr Valk u and Anton Berns1'4*

DivisionDivision of Molecular Genetics1, Division of MolecularMolecular Biology2, Department of ExperimentalExperimental Animal Pathology3 and Centre for BiomedicalBiomedical Genetics4, The Netherlands Cancer Institute.Institute. Plesmanlaan 121, 1066 CX Amsterdam,Amsterdam, The Netherlands.

"Correspondingg author

Thee retinoblastoma suppressor gene productt Rb has been assigned a critical role inn cell cycle regulation, the induction of differentiation ,, and inhibitio n of oncogenic transformation.. Germline inactivation of Rbb in mice leads to mid-gestational lethalityy with defects in erythropoeisis and neurogenesis.. This embryonic lethality prohibit ss the analysis of Rb function in selectedd cell types at later stages of developmentt or in the adult. Here, we describee the Cre-LaxP mediated somatic inactivationn of Rb in a subset of neuroendocrinee cells, including photoreceptorr cells. We observed neuro-endocrinee tumors of the pineal and pituitar yy gland. These tumors invariably showedd inactivation of Trp53. Remarkably, losss of Rb in photoreceptor cells does not leadd to retinoblastoma or any phenotypic changes,, not even when photoreceptor cells aree made deficient in Rb, pl07 and Trp53. Ourr results highlight the important differencess that exist in tumor susceptibilityy between mice and man and questionn the photoreceptor cell origin of humann retinoblastoma.

Keywords:: Retinoblastoma gene (Rb), Interphotoreceptorr Retinol Binding Protein (IRBP),, pi07, Trp53, photoreceptor cells, sporadicc tumors.

Introductio n n

Retinoblastomaa is a malignant tumor of the retinaa that is caused by inactivating mutations inn the retinoblastoma susceptibility gene (RBI) inn humans (Friend et al. 1986; Fung et al. 1987; Leee et al. 1987). Inheritance of a mutant RBI allelee is responsible for familial forms of this diseasee (Knudson 1971) and increases the risk off developing osteosarcoma and melanoma (Molll et al. 1997). Trilateral retinoblastoma (TRB)) is a rare variant of familial retinoblastomaa in which in addition to bilateral retinoblastoma,, tumors develop from the pineall gland (Jakobiec et al. 1977). RBI is also frequentlyy found mutated in sporadic cancer of thee lung, breast and bladder (Harbour et al. 1988;; Lee et al. 1988; Takahashi et al. 1991). Mutationall analysis of human tumors has shownn that components of the Rb pathway are foundd inactivated in virtually all tumor types (Weinbergg 1995). Reintroduction of Rb into RBIRBI deficient tumor cells suppresses tumor growthh in mice confirming its role as a tumor suppressorr (Riley et al. 1996). Rbb encodes a nuclear phosphoprotein that integratess extracellular signals to regulate cell cyclee progression (Weinberg 1995). The hypophosphorylated,hypophosphorylated, active form of Rb is complexedd to E2F/DP heterodimeric transcriptionn factors (Dyson 1998) and repressess transcription directly by binding to promoterss carrying E2F-binding sites (Zhang ett al. 1999). Cell cycle progression though Gl requiress functional inactivation of Rb by phosphorylation.. This is triggered by cyclin dependentt kinases (CDK) and their associated regulatoryy cyclins. Consecutive phosphorylationn of Rb by cyclin D/CDK4/6 andd cyclin E/CDK2 results in the release of boundd E2F/DP and promotes progression into S-phasee (Harbour et al. 1999). Another level of regulationn is mediated through the CIP/KIP and INK44 families of proteins. These proteins act too assemble as well as to inhibit the cyclin/CDKK complexes upon mitogenic signalingg (Sherr and Roberts 1999). Rb can also

49 9

ChapterChapter 4

activelyy promote differentiation by blocking Id22 or activating C/EBP (Chen et al. 1996; Lasorellaa et al. 2000). The Rb family of pocket proteinss comprises three functionally related members;; Rb, pi07 and pi30 with overlapping andd distinct functions (Mulligan et al. 1998) Of thesee RBI is most frequently found mutated in humann tumors but mutations have also been foundd in pi30 (Claudio et al. 2000). Micee homozygous for a null mutation in Rbl (Rb)(Rb) show embryonic lethality with defects in hematopoiesis,, neurogenesis and terminal lens differentiationn (Clarke et al. 1992; Jacks et al. 1992;; Lee et al. 1992; Robanus-Maandag et al. 1994).. Heterozygous Rb+/ ' mutant mice primarilyy develop melanotroph tumors of the pituitaryy gland and thyroid hyperplasia with losss of the wild type Rb allele (Jacks et al. 1992;; Hu et al. 1994). However, no retinoblastomass develop in Rb+I ' mice;

Chimericc mice composed of Rb-deficient and wildd type cells are viable and rapidly develop pituitaryy tumors and thyroid adenomas and showw a defect in terminal differentiation of lens epitheliall cells (Robanus-Maandag et al. 1994; Williamss et al. 1994b). While chimeric mice do nott develop retinoblastomas, Rb-deficient retinoblastss are impaired in their capacity to contributee to the adult retina (Robanus-Maandagg et al. 1994). The requirement for Rb functionn during retinal development is also supportedd by the phenotype of cyclin Dl null micee that suffer from retinal hypoplasia (Sicinskii et al. 1995). Cyclin Dl is highly expressedd in the embryonic retina and this correlatess with phosphorylation of Rb. The photoreceptorr cell degeneration in cyclin Dl nulll mice can be rescued by cyclin E when expressedd from the cyclin Dl locus or in a p27p27KIPKIP'''-deficient'-deficient background (Geng et al. 1999;; Geng et al. 2001). This indicates that the retinall hypoplasia in cyclin Dl null mice is a defectt in Gl cell cycle control that likely impingess on Rb regulation. Thee analysis of chimeras indicates that, in contrastt to man, inactivation of Rb in mice doess not lead to retinoblastoma and that additionall mutations are required for tumor formation.. One of these is the tumor suppressorr pi07, since chimeric mice partly composedd of cells lacking both pi07 and Rb

50 0

developp retinoblastoma (Robanus-Maandag et al.. 1998). Thesee tumors originated from the inner nuclear layerr (INL) of the retina and showed markers specificc for amacrine cell differentiation. However,, the majority of human retinoblastomass show characteristics of photoreceptorr cell differentiation, express markerss for cone- or rod photoreceptors and havee an intact phototransduction cascade suggestingg that they originate from the outer nuclearr layer (ONL) of the retina (Bogenmann ett al. 1988; Gonzalez-Fernandez et al. 1992). Alsoo the benign variant of retinoblastoma, retinocytoma,, shows photoreceptor cell differentiationn despite its location within the INLL (Margo et al. 1983; Gallie et al. 1999). Furthermore,, in mice, retinoblastoma with photoreceptorr cell characteristics could be inducedd by expression of SV40 large T-antigen (Tag)) using the Interphotoreceptor Retinol Bindingg Protein (IRBP) promoter (al-Ubaidi et al.. 1992; Howes et al. 1994a). Tag binds and inactivatess Rb family members, the p53 tumor suppressorr gene product (Trp53) and several otherr cellular proteins. The pleiotropic effects off Tag make it difficult to dissect the requirementt for the individual gene defects for retinoblastomaa formation in mice. Too further define the role of each of these geness in retinoblastoma formation we studied thee consequences of photoreceptor-specific inactivationn of Rb in conjunction with defects inn pi07 and Trp53. This is now possible using thee Cre-/ojcP and ¥\p-FRT methodology, which permitss the spatially and temporally controlled somaticc introduction of mutations in mice (Metzgerr et al. 1999). Utilizing these systems wee generated mice carrying a conditional mutationn in Rb and inactivated Rb in IRBP-expressingg cell types. Here we describe the consequencess of loss of Rb family members on photoreceptorr cell development and tumor formation. .

Results s

Cre-mediatedCre-mediated loss ofRb results in a null allele Homozygouss disruption of exon 19 of Rb in micee results in a null allele and embryonic lethalityy (Clarke et al. 1992). To address the functionn of Rb in photoreceptor cells we

TumorTumor formation in mice with somatic Rb loss

utilizedd the Cre-/axP system in order to accomplishh the tissue-specific inactivation of Rb.. Therefore we designed a targeting strategy thatt places loxP sites around exon 19 (i.e. RbRbFF'\'\ such that exon 19 can be deleted by Cre-mediatedd recombination (Fig 1 A) followingg a strategy similar to the generation of micee carrying the recognition sequences for FLPP recombinase around exon 19 (Vooijs et al. 1998).. Southern blot analysis performed on multiplee independent subclones showed that thesee clones displayed a hybridization pattern consistentt with targeting of the Rb locus and thee presence of both loxP sites surrounding exonn 19 (Fig 1 B). Homologous recombinant cloness were injected into C57B1/6 blastocysts andd transmitted through the germ line. RbF}9/F'9

micee were viable, fertile and showed no spontaneouss tumor predisposition similar to conditionall Rb mutant mice carrying FLP recombinasee recognition sequences at the same positionss (Vooijs et al. 1998). Deletionn of exon 19 via Cre-mediated recombinationn (Rb*19) is expected to result in a framee shift and premature termination of Rb. Too determine whether Cre-mediated deletion of exonn 19 results in a truncated allele we isolated primaryy keratinocytes from RbF19/F'9 mice expressingg Cre from the cytokeratin-14 promoterr (i.e. K14cre, M. Vooijs et. al., in preparation).. Primary keratinocytes express detectablee levels of all Rb family members (Martinezz et al. 1999). Immunoprecipitation followedd by western blot analysis using antibodiess directed against Rb showed that in RbRbFi9/Fi9.Fi9/Fi9. p}0Ti- keratinocytes, Rb but not pi077 could be readily detected (Fig 1 C). In contrast,, in keratinocytes derived from K14cre:RbK14cre:RbFI9/F,9FI9/F,9\\ plOt1' mice no Rb could be detected,, whereas pi07 was present in the samee extract. Extracts from both genotypes showedd comparable expression of pi30. Thus Cre-mediatedd deletion of exon 19 in vitro resultss in a failure to detect the expression of a full-lengthh Rb using an antibody that recognizess the carboxy-terminus. Too determine whether the RbA'9 allele behaves ass a null allele, we crossed RbFi9/F'9 mice with a germm line deleter strain (M. Vooijs and A. Berns,, unpublished). From these crosses RbRbA19/+A19/+ mice were obtained and intercrossed.

Inn the offspring we never found RbA,9/A'9 mice suggestingg that these mice died before birth. PCRR based genotyping of embryos at different dayss of gestation showed that Rb*/+, RbAim

andd RbAI9/A'9 mice respectively were found at thee expected frequency at day E13.5, whereas fromfrom day E 15.5 onwards no live RbA,9/A19

embryoss were found (Table 1). Thus, like Rb nulll embryos, RbAI9IM9 embryos fail to develop too term. Analysis of serial HE-stained sections fromfrom RbA19/A19 embryos at day E 13.5 showed phenotypicc aberrations reminiscent of a null mutationn in Rb: pyknotic cell death in the peripheral-- and central nervous system (Fig 1 D-G),, defective erythropoeisis and aberrant lenss development (not shown). Together these findingss show that the Rb conditional allele actss as a wild type Rb allele and that Cre-mediatedd excision of exon 19 in mice phenocopiess a null allele.

ConditionalConditional inactivation ofRb in the retina Too investigate the role of Rb in retinoblastoma developmentt we placed the gene encoding Cre recombinasee under control of the human IRBP promoterr to direct Rb inactivation to the photoreceptorr cells. IRBP expression in transgenicc mice has been reported to start at dayy E13 and to continue after birth in postmitoticc rod- and cone photoreceptor cells off the outer nuclear layer (ONL) and in pinealocytess of the pineal gland (Liou et al. 1990;; al-Ubaidi et al. 1992). We generated two liness of IRBP-cre transgenic mice: IC-64 and IC-47,, respectively with a different transgene copyy number (Akagi et al. 1997). To determine thee pattern of Cre recombinase activity in thesee mice, we generated IRBP-cre:AcZL doublee transgenic mice. The AcZL reporter line allowss in situ identification of Cre activity by excisionn of a floxed STOP cassette and concomitantt activation of lacZ (Akagi et al. 1997).. In both transgenic lines Cre activity couldd be detected in the retina and in cells from thee pineal gland. Within the retina a mosaic patternn of clusters of X-gal stained cells was foundd in both the peripheral as well as in the centrall part of the ONL at 2 weeks of age (Fig 22 A,B). X-gal staining was only seen in the outerr segments (OS), the ONL and outer plexiformm layer (OPL) all of which harbor

51 1

ChapterChapter 4

»\ »\ probe- B B

tr r

m' m'

44 5kb

100 kb

zz cr

EB B 7kb b

100 kb

ÜJ J

E E DC C

probe-C C

TE-- -y^ ^

1 1

--2 2

-. . Rb b

p107 7

HH t l p130

a11 a2 d c2

R 6 3 ' - / o x P ^ ^

R 6 " " » --

RbRb5--loxP*. 5--loxP*.

R b » " » --

„. . .

EcoRV-probe- C C

. .

-- 10 kb -- 7 k b

-- 10 kb

-- 4.5 kb

EcoR\/Nhe\-EcoR\/Nhe\- probe-B

Figuree 1. Generation and charaeterization of the conditional Rb allele. (A) structure of the mouse Rb locus and the targeted LoxP modified Rb

locuss (RbF1P). The presence of loxP sites disrupt Nhel and EcoRW restriction sites, and were used to identify targeted clones. Nh, Nhel; RV,

EcoRV;EcoRV; RI, EcoSl, (B) Southern blot analysis on independent 6TG-resistant subclones carrying both lox? sites. Upper panel shows detection of the

3'-loxP3'-loxP site using probe C on EcoKV digested ES cell DNA. A wild type 7 kb fragment and a novel 10 kb fragment for the 3'-/oxP insertion are

present.. Lower panel shows identification of the 5'-/orP insertion using probe B after an EcoRVNhel double digest in targeted clones resulting in a

100 kb fragment for the I ox? modified Rb allele and a 4.5 kb fragment for the wild type allele. (C) Cre-mediated deletion of exon 19 results in loss

off Rb protein in primary cells. Sequential immunoprecipitation followed by western blot analysis on extracts from primary keratinocytes from

RbRbFI9IFFI9IF ''99;; pIOT' (lane 1) and Kl4cre:RbF19'F19; plOf" (lane 2) with Rb, pl07 and pl30 antibodies. Cre-mediated deletion of Rb in primary

keratinocytess (lane 2) results in undetectable levels of Rb under conditions where p!07 or pl30 can be readily detected. Rb ; pIOT

keratinocytess express Rb and p!30 but not pl07 (lane I). (D-G) The RbLl9m9 allele acts as a null allele. Shown are HE-stained sections from

E13.55 day littermates showing detail of trigeminal ganglia (TG, D and E) and dorsal root ganglia (DRG, F and G) of Rb A (D and F) and

Rb*'Rb*'mm''99 (E and G) mice. Note the massive increase in pyknotic cell death (arrow) in TG and DRG from Rbsi9s'9 mice. Original magnification D-

C,20x. .

Tablee 1.» Embryonicc lethality of RbA/9/A/i i

genotype e

## mice

%% found

%% expected

Adult t

A19/+ A19/+

22 2

50 0

50 0

M9/M9 M9/M9

----

25 5

+/+ +

22 2

50 0

25 5

micee from a RbAlw+ K.. RbA19/+ intercross

E13.5 5

A19/+ A19/+

18 8

56 6

50 0

&19/A19 &19/A19

9 9

28 8

25 5

+/+ +

5 5

16 6

25 5

E15.5 5

A19/+ A19/+

9 9

47 7

50 0

A19/A19 A19/A19

----

25 5

+/+ +

12 2

53 3

25 5

Tablee 1. Onset of embryonic lethality o/Rb&imJ9 embryos. RbAI<" mice were intercrossed and the genotype of the offspring was analyzed by PCR

onn yolc-sac or tail DNA. In 44 adult mice analyzed no homozygous Rb*1 mice were found. Live Rb embryos were readily detected at

dayy E13.5 (9/32, 28%) but absent at E15.5 (n=21). The RbiJ'9m9 embryos had a paler appearance and were smaller than their normal littermates.

52 2

TumorTumor formation in mice with somatic Rb loss

photoreceptorr cells and no staining was seen in otherr cell types in the retina. Too determine whether Cre was active in both rodd and cone-photoreceptor cells we made use off the fact that FVB/N mice suffer from an inheritedd retinal degeneration (rd) resulting in thee complete loss of rod but not cone photoreceptorr cells by 4 weeks of age (Carter-Dawsonn et al. 1978). Therefore, after degenerationn of rods from the retina by rd remainingg cone cells should retain LacL staining.. As expected IRBP-cré^-AcZL mice off 2 weeks and older showed a progressive degenerationn of rods from the ONL and a concomitantt reduction in the number of X-gal stainedd cells. However, some photoreceptor cellss still stained at an age of several months demonstratingg that IRBP-Cre was also active inn cone-photoreceptor cells (Fig 2C) (Akagi et al.. 1997). We also monitored Cre activity in thee pineal gland. IC-64:AcZL mice showed stainingg of virtually all pinealocytes (Fig 2 D) whereass IC-47: AcZL showed a more scattered X-gall staining (not shown). To determine the onsett of Cre expression in the neuroretinas of IC-47IC-47 mice we used immunohistochemical stainingg for Cre on tissue sections from embryoss at various times of gestation. Cre expressionn in IC-47 retinas was first observed att day E14.5 in the lower part of the ventricularr layer and continued to increase up too birth, showing a mosaic expression pattern throughoutt the ONL (Fig 2 E and not shown). Onee week after birth, photoreceptor cells in thee peripheral and in the center of the retina expressedd Cre in mosaic pattern reaching the OPLL (Fig 2 F,G). Cre expression in these cells continuedd for at least another two weeks after birthh (Fig 2 H). In addition a very small numberr of cells within the OPL near the INL showedd Cre expression presumably rods that aree still migrating. Line IC-64 did not show expressionn at day E 14.5 and started to express aroundd birth (not shown). The onset and expressionn characteristics of IC-47 mice are comparablee with that reported for other IRBP transgenicc constructs (al-Ubaidi et al. 1992; Yokoyamaa et al. 1992).

RbRb Junction is not required for rod or cone photoreceptorphotoreceptor differentiation Losss of Rb function in mice has been reported too result in aberrant proliferation followed by apoptosiss in several tissues including photoreceptorss of the retina (Howes et al. 1994b;; Robanus-Maandag et al. 1998). To investigatee the consequences of loss of Rb functionn on photoreceptor cell development andd tumor formation we generated IC-47:Rb47:RbF19/FF19/F''99 mice and compared the retinas of thesee mice with non-transgenic littermates at PND77 when the majority of photoreceptor cellss are terminally differentiated (Young 19855 a). A histological examination of retinas fromm control versus IC-47:RbFI9/F'9 mice did nott reveal any differences in cytoarchitecture (Figg 3 A), nor in proliferation as determined by immunohistochemicall staining for BrdU (Fig 3 C).. At PND7 a limited but comparable number off BrdU stained cells was observed in the peripherall ONL retinas from IC-47:RbF19IF19

andd wild type mice (not shown) where proliferationn ceases at PND11. During normal retinall development very few photoreceptor cellss in the ONL are eliminated by apoptosis. Inn the ONL of IC-47:RbFI9/F,g mice analyzed at PND77 no evidence was seen for increased apoptosiss compared to controls (Fig 3 D). In contrastt tunel labeling readily identified apoptoticc cells in the INL of control and IC-47:Rb47:RbFI9IFFI9IF ''99 mice at PND7 reflecting histogenic celll death that normally occurs at this stage of developmentt (Young 1984). Apoptosis in the INLL decreased after this time point and could nott be detected in the INL or the ONL of controll and IC-47 :RbFl9IFl9 mice three weeks afterr birth (not shown). A further comparison off retinas from transgenic and non-transgenic RbRbF19IF19F19IF19 mice at day E 14.5, PND1, PND7 and att three weeks after birth did not reveal any obviouss differences in terms of morphology, proliferationn or apoptosis (not shown). To investigatee whether Rb-deficient were being removedd and replaced by non-IRBP-Cre expressingg cells during postnatal development wee determined if Cre was still expressed in the retinass from IC-47 \RbFl9IFl9 mice. Noo major differences were found in the number orr location of IRBP-Cre expressing cells

53 3

ChapterChapter 4

f f

hi hi aa »

Figuree 1. Activity and expression analysis oflRBP-Cre. In situ identification of Cre-mediated recombination using the AcZL reporter transgene.

(A)) X-gal staining in the peripheral retina and (B) posterior retina of IC-47:AcZL'J' mice and (C) on the rd/rd background at 2 months of age. (D)

X-gall staining in the pineal gland of IC-64:AcZL mice. Note the typical radial pattern of X-gal stained cells in the ONL of the retina and

persistencee of blue stained cells in rd/rd retinas. Immunohistochemical staining for Cre in IC-47 retinas. (E) at day EI4.5 (F), at PND7 in peripheral

andd (G) posterior retina of IC-47:RbFlm mice and (H), at 3 weeks after birth. Sections are counterstained with nuclear fast red (A-C) and HE (D-

H).. Original magnification is A-D, lOx; and E-H, 20x.

'' t9»'MJV0

-<-.. T - t -

II & 2 0>> O) O) T -- 't— T—

u.. u. u_ -QQ -Q .Q KK K CC

TT F 'INL 'INL

OPL OPL

ONL ONL

OS OS

F19 F19 Rb Rb

Rb Rb 419 419

Cre:: +

Figuree 3. The effect o/Rb loss in the ONI of IC-47 :Rbmr" mice at PND 7 (A) HE-stained section of the retina. (B) Cre expression in the ONL (C)

BrdUU incorporation and (D) Tunel labeling. Note apoptotic cell death in the 1NL and absence of Tunel or BrdU positive cells in the posterior ONL.

Identicall results were obtained with all genotypes (see text). Counterstain is haematoxilin (B,C) or methyl-green (D). (E) Expression of Cre results

inn the deletion of Rb in ONL of the retina at PND1. Micro-dissection PCR on ONL of IC-47:Rbf"'\ non-transgenic Rb""m retina and RbF"" A"

control.. Note significant deletion of Rb in ONL photoreceptors in transgenic but not in non-transgenic £6™"" mice. INL (Inner nuclear layer),

OPLL (Outer plexiform layer), ONL (Outer nuclear layer), OS (outer segments). Arrows indicate tunel positive cells Original magnification A-D,

20x. .

betweenn the retinas from IC-47:RbFIW+ and IC- performed on the ONL of IC-47:RbF

47:RV"47:RV"yy"" ,,''yy mice one week after birth indicatingg that no selection occurs against Cre expressingg cells that carry a bi-allelic Rb conditionall allele (Compare Fig 2 G and 3 B). Too ascertain that under conditions of reporter genee switching, Rb was also inactivated by Cre-mediatedd recombination, we performed a PCRR analysis on the ONL from normal and transgenicc retinas obtained by micro dissection fromfrom HE-stained sections. This analysis

mice e showedd that fragments indicative of exon 19 excisionn by Cre (yielding Rb& '9) could be readilyy amplified (Fig 3 E and not shown). Too investigate the possibility that the absence off pathology in Rb-deficient photoreceptor cellss is caused by functional compensation of thee Rb related family member pi07 (Robanus-Maandagg et al. 1998), we generated mice lackingg both Rb and pi07 in photoreceptor cells.. P707-deficient mice are viable and show

54 4

TumorTumor formation in mice with somatic Rb loss

noo phenotypic aberrations on a mixed FVB/1299 genetic background (J-H Dannenberg, unpublishedd observations).As expected, the retinass of /?/07-deficient mice showed comparablee levels of proliferation and apoptosiss as in wild type mice (not shown). Remarkably,, deletion of both Rb and pl07 in #?#P-expressingg cells of the retina (i.e. IC-47:Rb47:RbFI9/FFI9/F''99;; plOT1) did not lead to any changess in proliferation or apoptosis (not shown).. Accordingly, Cre expression in these retinass was comparable to control JC-47:Rb47:RbFI9/+FI9/+ ;; plOT1' retinas at PND7 (not shown).. Thus we conclude that photoreceptorss lacking both Rb and pi07 contributee and persist in the retinas of adult micee and are apparently normal.

RbRb loss in IRBP-expressing cells leads to neuro-endocrineneuro-endocrine tumors Too investigate the consequence of somatic Rb inactivationn on tumor formation in the retina or thee pineal gland we examined a cohort of IC-6464..RbRbFi9!F,9Fi9!F,9 ( n = 2 6) md IC-47:RbF,9IFi9 (n=33) mice,, bi-weekly for the development of tumors.. These mice with an average life span off 384 days did not develop retinoblastomas or pineall gland tumors but succumbed to pituitaryy gland tumors instead (Table 2., Fig 5 A).. These tumors arose from the anterior lobe off the pituitary gland (Fig 4 A) as well as from from melanotrophh cells of the intermediate lobe (Fig 44 C). Sometimes both tumors occurred within onee animal (Fig 4 B). Melanotroph tumors in thesee mice were indistinguishable from those thatt spontaneously develop in Rb+ '~ mutants or inn mice with pituitary-specific Rb loss (Robanus-Maandagg et al. 1994; Vooijs et al. 1998).. Anterior lobe pituitary tumors could be classifiedd histologically into two groups, a ratherr monomorph cavernous type of tumor withh a tendency to hemorrhage and a second consistingg of highly pleomorphic tumor cells demonstratingg anisonucleosis, hyperchromatic nucleii and poikilonucleosis sometimes with concomitantt mineralization. Infrequently, both typess occurred within one and the same tumor (Figg 4 D). To further characterize the origin of thee anterior lobe pituitary tumors we used immunohistochemistryy against 5 different

pituitaryy hormones expressed in the normal anteriorr lobe but did not find any evidence for hormonee production in nine tumors tested, withh the exception of the intermediate lobe pituitaryy tumors that were positive for a-MSHH (not shown). Immunostaining against the neuro-endocrinee marker Synaptophysin was oftenn positive for the anterior lobe tumors indicatingg some degree of neuro-endocrine differentiationn (Fig 4 E).

RbRb and Trp53 loss synergize in neuro-endocrineendocrine tumor formation Manyy human tumors show mutations in both RBIRBI and TP53 pathways (Hanahan et al. 2000) andd in mice mutations in Trp53 and Rb stronglyy cooperate in tumor development in a widee range of tissues (Williams et al. 1994a; Marinoo et al. 2000). To investigate if Trp53 losss collaborates with Rb loss on anterior lobe pituitaryy tumor formation we crossed IC:RbIC:RbFI9/FI9FI9/FI9 mice with Trp53 mutant mice (Donehoweretal.. 1992). IC:RbF19/F'9; Trp53^' micee (n=61) developed tumors with a significantlyy reduced mean latency of 267 days (Figg 5 A). In addition to anterior lobe tumors, IC:64:RbIC:64:RbF19/F19F19/F19;; Trp53+/- mice also developed tumorss originating from the pineal gland (Table 2.,, Fig 4 F-K). Importantly, pituitary and pineall tumors did not arise spontaneously in Trp53Trp53+I+I '' mice that succumbed from tumors withh a mean latency of 517 days (Fig 5 A). Histologicall examination showed that pinealoblastomass locally invaded the brain and showedd a highly undifferentiated aggressive andd anaplastic cytological appearance and a highh degree of pyknotic nuclei (Fig 4 F,G,I). Withinn these tumors occasionally regions of well-differentiatedd areas could be found resemblingg the Flexner-Wintersteiner rosettes characteristicc for human pinealoblastoma and retinoblastomaa and indicative for photoreceptorr cell differentiation (Fig 4 H). Immunostainingg showed that pineal tumors expressedd the neuro-endocrine marker Synaptophysinn but not the glial marker GFAP (Figg 4 J,K). In addition tumors showed focal expressionn of IRBP and S-antigen (not shown) bothh expressed in the normal pineal gland (Blackshaww et al. 1997).

55 5

ChapterChapter 4

P„.. v v

' -v-.r^- a a

Figuree 4. Hislopalhology in ICRb ; Trp53' mice. Pituitary gland tumors (A-E) Histological sections of a pituitary gland. (A) anterior lobe

tumorr (B) a bi-pituitary tumor and (C) intermediate lobe tumor. (D) A biphasic anterior lobe tumor with a highly pleomorphic hyperchromatic

componentt with anisonucleosis and poikilonucleosis . (E) Synaptophysin positive immunostaining in anterior lobe tumors. Pineal gland tumors, at

loww magnification (F) showing location of the pineal gland tumor and invasion into the surrounding brain parenchyma(G). (H) Flexner-

Wintersteinerr rosettes in pineal gland tumors. (I) HE-stained section of pineal gland tumor and (J) Synaptophysin staining and (K) GFAP staining.

Remnantss of the normal pineal organ are visible that stain with Synaptophysin but not GFAP (arrow). Magnification H, lOOx; A,C, 40x; D, E, G,

20x;; I-K, 10x;B;2.5x.

LossLoss of heterozygosity of Trp53 in neuro-endocrineendocrine tumors with Rb loss Thee significantly reduced median survival (209 dayss vs. 389 days, p-value < 0.001) of lC:RblC:RbF,9,FF,9,F''99;; Trp53+/~ mice compared to \C:Rb\C:RbFl9IFFl9IF ''99 mice indicate that Rb and Trp53 actt synergistically to suppress tumor formationn in the pituitary and the pineal gland. Too further investigate this, we determined whetherr tumors had undergone loss of heterozygosityy of the wild type Trp53 allele andd Cre-mediated deletion of exon 19 of Rb. Bothh in anterior lobe tumors (10/10) and in pinealoblastomass (4/4) we found a major reductionn or complete absence of fragments specificc for the wild type Trp53 allele as examinedd by allele-specific PCR (Fig 5 B,C). In supportt of this, tumor formation was further acceleratedd in IC-64:RbFl9IFI9; Trp53-'' mice thatt developed pineal gland tumors (8/13) and

aa small number of anterior lobe tumors (2/13) withh an average latency of 88 days (Table 2 andd Fig 5 A). Remarkably, no retinoblastomas developedd in IC;Rb Trp53"Trp53" mice that alsoo lacked one (7/13) or both copies of pi07 (2/13),, respectively. IC-64:RbF,9IF,9\ TrpSï'-micee spontaneously developed several tumor typess also observed in a large cohort of non-transgenicc Trp53''~ mice (n= 56) such as haemangiosarcomas,, teratocarcinomas and thymicc lymphomas (Table 2) (Donehower et al.. 1992) As expected anterior lobe tumors and pinealoblastomass showed Cre-mediated loss of RbRb (Fig 5 B and C and not shown). The earlier appearancee of pineal gland tumors and anterior lobee tumors in IC;RbF,9IF'9; Trp53-'- mice furtherr supports the oncogenic collaboration betweenn Rb and Trp53 mutations in these tumors. .

56 6

TumorTumor formation in mice with somatic Rb loss

Howeverr since the median tumor free survival off Trp53-'- versus IC;RbFl9IF19; Trp53 ̂ mice didd not markedly differ (91 days vs. 86 days) wee conclude that these mice primarily succumb too tumors that do not depend on Rb loss. We alsoo investigated whether p!07 loss contributedd to the development of pineal gland orr anterior lobe pituitary tumors. No effect wass seen of pi07 loss in lC:RbFI9/Fi9; plOT1-(n== 13) mice on the rate of tumor development orr on the tumor spectrum (not shown) consistentt with the fact that we did not find losss of the pi07 wild type allele in tumors fromm \C:RbF19IF19\ Trp53+I '; plOf' plOf' mice (Fig 5 B,C). .

Discussion n

ConditionalConditional mutagenesis Thee generation of knockout mice has provided insightt into the role of Rb in specific developmentall processes as well in suppressingg cancer in mice (reviewed in Vooijs ett al. 1999). However, the embryonic lethality off Rb null mice prohibits the phenotypic analysiss of Rb-deficiency in adult mice. Unlike humanss where RBI heterozygosity invariably leadss to retinoblastomas, in mice this leads to intermediatee lobe pituitary tumors and thyroid hyperplasiaa with high incidence (Hu et al. 1994).. Likely, Rb+I~ mutant mice succumb from thesee tumors before the onset of other malignanciess in which RBI loss is also found in man.. This is illustrated by the analysis of Rb+I ' ;; Trp53~'~ mice that develop a wide spectrum of primarilyy neuro-endocrine tumors with loss of thee wild type Rb allele (Williams et al. 1994a). However,, such compound mutant mice are difficultt to maintain and develop a much wider spectrumm of tumors, many of which show no involvementt of Rb. This has prohibited a detailedd analysis on the role of Rb in tumor onsett and progression of specific tumor types inn mice. Conditional somatic mutagenesis circumventss this problem as it permits the introductionn of mutations in a specific subpopulationn of cells. Furthermore, since one cann choose conditions in which gene inactivationn is occurring in only a small fraction off the cells it is possible to better mimic the onsett of sporadic cancer in man.

RbRb function in photoreceptors Too specifically address the role of Rb in photoreceptorr cell development and tumorigenesiss we disrupted Rb in cone- and rodd photoreceptors of the retina using IRBP drivenn Cre recombinase expression. We found thatt Cre recombinase is expressed and active in rod-- and cone photoreceptors of the ONL and inn the pineal gland. The striped X-gal staining patternss we observed in the retinas of adult IRBP-cre:AcZLIRBP-cre:AcZL transgenic mice indicated that switchingg already occurred in the progenitor cellss leading to lineage transmission of the activatedd galactosidase to their descendents (Williamss et al. 1992). Expression of Cre was firstfirst observed in the ventricular neuroretina at dayy E14.5 and continued to be expressed in a similarr pattern until birth. In the first week afterr birth Cre was robustly expressed throughoutt the entire ONL and expression continuedd at least for another two weeks (Fig 2).. We conclude that Cre expression in our micee resembles the normal pattern of endogenouss IRBP expression (Liou et al. 1994). .

Inactivationn of Rb from day E 14.5 onwards in IRBP-expressingg cells did not lead to any pathologyy in de retina. The time of onset and cell-typee specificity of Cre expression reportedd here indicates that a significant proportionn of rods as well as cones have becomee /?£-deficient during their genesis and persistedd in the adult retina (Carter-Dawson et al.. 1979; Young 1985b; Young 1985a). Thus Rbb function is not required to maintain the postmitoticc state of photoreceptor cells nor didd bi-allelic Rb loss predispose to retinal tumorss in mice. We investigated whether the lackk of pathology could be attributed to functionall compensation by the Rb family memberr pi07 (Robanus-Maandag et al. 1998). Ourr analysis shows that inactivation of both Rbb and pi07 does not affect the proliferation orr terminal differentiation of cone- or rod photoreceptorr cells in the postnatal retina. No retinoblastomass developed in compound IC-RbRbF19/F19.F19/F19. p W T l - m i c e Q u r f m d i n g s c o n t r a st

withh the occurrence of retinoblastoma in IRBP-TagTag transgenic mice, the depletion of Rb (pl07)-deficientt photoreceptor cells in

57 7

ChapterChapter 4

Tablee 2. Tumor spectrum in mice with somatic Rb loss in IRBP-expressing cells

RbRb ''"""">

Meann lifespan days (+/-sem)

Anteriorr Lobe Tumor (%)

Melanotrophh Tumor (%)

Bi-pituitaryy Tumor (%)

Pituitaryy Tumor (%)

Pinealoblastomaa (%)

Otherr Tumors (%)

Noo Tumors found (%)

Numberr of mice

IC-64 IC-64

350(137) )

15(58) )

1(4) )

2(8) )

1(4) )

0 0

6(23) )

1(4) )

26 6

IC-47 IC-47

411(107) )

10(30) )

12(36) )

3(9) )

5(15) )

0 0

K3) ) 2(6) )

33 3

IC-64IC-64 +

IC-47 IC-47

384(124) )

25(42) )

13(22) )

5(8) )

6(10) )

0 0

7(12) )

3(5) )

59 9

Trp53Trp53 "

IC-64 IC-64

264(123) )

32(63) )

1(2) )

0 0

4(8) )

8(15) )

6(11) )

5(10) )

51 1

Trp53Trp53 "

IC-47 IC-47

278(128) )

5(50) )

0 0

3(30) )

1(10) )

0 0

1(10) )

0 0

10 0

Trp53Trp53 ''

IC-64 IC-64

888 (29)

2(15) )

0 0

0 0

0 0

8(62) )

111 (85)

2(15) )

13 3

Tablee 2. Summary ofhistopathotogical findings in IC-Rb ' ; Trp53 mutant mice. Mice were examined macroscopically and microscopically for

thee presence of tumors. Incidence of each lesion is expressed as the percentile of the total number of animals in a specific group. The mean

lifespann and the mean error are indicated for each group of animals. Some animals showed evidence for the presence of both a melanotroph as

welll as an anterior lobe tumor (bi-pituitary tumor). Pituitary tumor indicates macroscopically a pituitary tumor was seen but the type was not

confirmedd histologically. A group of 56, Trp53" mice were followed which developed tumors with an average latency of 96 (+/- 35) days. Mice

developedd haemangiosarcomas (69 %), lymphosarcoma (40%) and teratocarcinoma (10%). Trp53 ' mice often had more than one different tumor.

00 100 200 300 400 500 600 700 800 900

dayss in experiment

F igures.. Tumor incidence in IRBP-cre (IC) mice with conditional Rb inaciivation and Trp53 mutation. (A) Graph summarizing the tumor-free

survivall of IC-RbFt9,F'9, IC-RbFI9tF19; Trp53^~, IC-RbFI9/F'9; Trp53'~and non-transgenic Trp53'' and Trp53* mice. The median tumor free survival

(T,«.)) was for IC-Rb"91"9 (389 days), for IC-RbFmFI9;Trp53* (209 days). RbFI9fF'9; Trp53' (86 days), Trp53-i- (91 days) and 516 days for Trp53'

mice,, respectively. Note significant acceleration of tumor development in mice with combined mutations in Rb and Trp53. Number of mice used in

eachh group is indicated. (B) Representative PCR analysis to determine the loss of Rb (lanes \-4),pl()7 (lanes 5-8) and Trp53 alleles (lane 9-12) in

anteriorr lobe tumors (ALT) and normal tissue (N) from IC;RbFI9FI9;pI07"; Trp53' mice. Cre-mediated loss of Rb is observed in tumors but not

normall tissue and loss of the Trp53 but not thepl07 wild type allele is only seen in tumors. (C) Representative PCR analysis to determine the loss of

RbRb (lanes 1-4), pW7 (lanes 5-8) and Trp53 alleles (lane 9-12) in pinealoblastomas (PB3) versus normal tissue (N). Pineaoloblastomas show loss of

RbRb and Trp53 but not pl07 in IC-Rb mF,9\ pJOf'; Trp53H' mice. Right panel shows Cre-mediated Rb loss in pinealoblastoma (PB4) but not in

haemangiosarcomaa (HS) from the same IC-RbF19,F,9\ Trp53" animal.

58 8

TumorTumor formation in mice with somatic Rb loss

Rb'Rb' ~/Rb+ chimeric mice and the development off retinal dysplasia in Rb+/ '; plOT1' or Rb+I '; Trp5S'Trp5S'!! '' mutant mice (al-Ubaidi et al. 1992; Williamss et al. 1994a; Lee et al. 1996; Robanus-Maandagg et al. 1998). Thus importantt differences exist between somatic Rbb loss in our model and previously described models. . Thesee differences can be explained in several ways:: i. Rb loss in our model occurs in a very limitedd number of cells. Our data show that Cree is robustly expressed and mediates switchingg of a reporter substrate and deletion off Rb in the ONL of the retina the first week afterr birth (Figs 2 and 3). Because the PCR analysiss performed to determine Rb deletion in thee retina is not quantitative it is difficult to estimatee the actual number of ^-deficient cells presentt at birth. We recently reported that Cre-mediatedd recombination of the RbF19 allele inin vivo is extremely efficient when compared withh other conditional alleles in the same cell (Vooijss et al. 2001). Therefore, we expect that Cree activity as measured by the reporter substratee underestimates the number of Rb-deficientt cells at the various time points. Undoubtedly,, loss-of-Rb function will occur at aa much higher frequency than the spontaneous losss of the wild type allele in Rb+I '; plOT1' micee (Mulligan and Jacks 1998). Consequently,, it is extremely unlikely that the lackk of any pathology is due to an extremely loww number of Rb-deficient photoreceptor cells,, ii . If Rb inactivation occurs relatively late,, sufficient Rb may remain to assure terminall differentiation and prevent tumor formation.. The observation that the timing of RbRb inactivation can dramatically affect the cellularr consequences of Rb loss has been documentedd for cortical neurons where early losss of Rb results in apoptosis while late inactivationn does not (Slack et al. 1998). In this respect,, important differences exist between functionall inactivation of Rb via viral oncoproteinss as opposed to Cre-ZoxP mediated gene-inactivation.. In IRBP-Tag mice, Tag will bindd and inactivate Rb at the onset of IRBP expressionn (i.e. at day E 14.5). In contrast, in IRBP-creIRBP-cre mice, the resident Rb mRNA and Rb proteinn will persist for some time after Cre-mediatedd inactivation of Rb has occurred.

Sincee Rb is a relatively stable protein (Mihara ett al. 1989) cells may have to undergo a substantiall number of divisions before becomingg Rb-deficient. This could explain the normall development of Rb-deficient cones that becomee postmitotic already around the onset off Cre expression. However, rod photoreceptorr cells then will still go through a majorr wave of expansion (Carter-Dawson and LaVaill 1979; Young 1985a). Hence, a significantt fraction of rod photoreceptor cells wil ll be Rb-deficient around birth. In our view, thiss justifies the conclusion that terminal differentiationn of rods can occur in the absence off functional Rb and/or pi07 protein. Therefore,, it is unlikely that loss-of-Rb blocks photoreceptorr cell differentiation, which in turnn could foster retinoblastoma development. iii .. Additional mutations are required to elicit photoreceptorr cell tumors. Our results show thatt loss of Rb, pl07 and Trp53 in photoreceptorr cells does not lead to tumors whereass expression of Tag does (al-Ubaidi et al.. 1992; Howes et al. 1994a) Formally, we cannott rule out that other functions of Tag mayy be required for tumor development. Amongg these may be inactivation of the Rb familyy member pi30 not affected in our experiments,, iv. The IRBP-expressing cell is nott the precursor cell of the retinoblastoma tumorr cells. Human retinoblastomas are thoughtt to originate from a progenitor capable off photoreceptor differentiation or Muller cell differentiationn (Gonzalez-Fernandez et al. 1992;; Nork et al. 1995). Interestingly lineage markingg has shown that both cell types may sharee a common precursor in the rodent retina andd that competence is maintained even after terminall mitosis (Cepko et al. 1996 and referencess therein). Case reports on benign retinoblastomaa suggest that tumors may originatee from the inner nuclear layer while showingg resemblance with photoreceptor cells (Margoo et al. 1983; Gallie et al. 1999). In keepingg with this, chimeric mice deficient for bothh Rb and pi07 develop retinoblastomas originatingg from the inner- but not the outer nuclearr layer (Robanus-Maandag et al. 1998). Iff this is the case, no tumors can be expected, ass the INL cells that give rise to retinoblastoma inn chimeric Rb1 \pl0T'iRb^\pl0T ̂ mice do

59 9

ChapterChapter 4

nott express IRBP and therefore, have not lost Rbb in our system. Thus it is possible that IRBPP is not expressed in the retinoblastoma precursorr cell but that expression of IRBP frequentlyy seen in human retinoblastoma reflectss the capacity of tumor cells to transdifferentiate.. This is currently our favorite explanationn and in accordance with the detailed analysess performed in chimeric mice (Robanus-Maandagetal.. 1998). Finally,, it has been reported that multiple loci inn the FVB genetic background suppress retinoblastomaa formation in mice (Griep et al. 1998).. These effects are predominantly caused byy a homozygous mutation in rod cGMP-phosphodiesterasee mutation that leads to photoreceptorr cell degeneration (Bowes et al. 1990).. To minimize the effects of genetic backgroundd on tumor predisposition in our micee that are of a mixed 129 Ola/FVB background,, mice were also kept heterozygous forr the rd allele. However, the presence of otherr modifier alleles from FVB or Ola 129 may alsoo contribute to the absence of phenotypic aberrations.. Currently, we cannot exclude this possibility.. Several of the explanations mentionedd above can be experimentally tested andd this will be focus of future work.

RbRb and Trp53 loss synergize in tumor formation formation Despitee the lack of retinoblastomas in mice withh photoreceptor cell specific inactivation of Rb,Rb, mice developed pituitary gland and pineal glandd tumors. The development of pineal gland tumorss is of interest as pinealoblastomas occur att low frequency as second primary tumors in patientss with hereditary retinoblastoma. This syndromee is referred to as trilateral retinoblastomaa (TRB) and also includes other intracraniall malignancies of primitive neuro-ectodermall origin (Bader et al. 1982 and referencess therein). The close phylogenetic relationshipp between the pineal gland and the photoreceptorr cells of the retina has led to the suggestionn that these organs share a common precursor,, from which retinoblastoma may arise.. Pinealoblastomas also arise in IRBP-Tag transgenicc mice and in Rb+I '\ Trp53'!' mice (Howess et al. 1994a; Williams et al. 1994a). Thee high incidence of pineal gland tumors in Rb

conditional/7>7?555 mutant mice and the absence off retinal tumors, illustrates a fundamental differencee with the human condition; i.e. RBI heterozygosityy in man invariably leads to retinoblastomaa but rarely to pineal tumors, whereass loss of Rb and Trp53 are rate-limiting stepss in the development of pinealoblastoma butt not for retinoblastoma formation in mice. Remarkably,, TP53 is rarely found mutated in humann pinealoblastoma (Tsumanuma et al. 1995)) but invariably lost in the pineal tumors wee have studied. In view of this it would be worthh re-examining whether other TP53 pathwayy components are mutated in pineal glandd tumors in man. IRBPP targeted Rb loss also resulted in the developmentt of anterior lobe tumors in mice. Thee development of these tumors was dramaticallyy accelerated in the Trp53 heterozygouss background and consistently showedd loss of the remaining Trp53 wild type allele.. The non-producing pituitary gland tumorss we found differed from the spontaneouss anterior lobe tumors observed in a fractionn of Rb+/ ' mice (Nikitin et al. 1999). As Rbb loss is a rate-limiting step in pituitary tumorr development in mice it is possible that Cre-/oxPP mediated bi-allelic Rb loss results in moree advanced tumors that are less well differentiated.. Interestingly, Trp53 loss is not involvedd in the development of intermediate lobee tumors in mice (Williams et al. 1994a) M. Vooijss and A. Berns, unpublished). The acceleratedd development of anterior lobe tumorss but not intermediate lobe tumors in micee with combined loss of Rb and Trp53 furtherr supports this. RBI and TP53 gene inactivationn has not been found to play an importantt role in the pathogenesis of common typess of human pituitary tumors (Levy et al. 1994;; Woloschak et al. 1994). Pituitary adenomass in humans often show silencing of pl6'"pl6'"k4ak4a by methylation instead (Woloschak et al.. 1997). Thus different components of the samee signaling pathways may be affected in murinee versus human tumors. Remarkably,, humans are the only species that spontaneouslyy develop retinoblastoma. This is invariablyy associated with loss of Rb function. Retinoblastomaa development in mice clearly requiress additional mutations. This may be

60 0

TumorTumor formation in mice with somatic Rb loss

relatedd to a different physiology and compositionn of the human versus the rodent retina.. However, it is also possible that in the humann retina one-or-more signaling pathways aree partially deficient or constitutively active makingg the retina highly tumor prone during a particularr developmental window. In such a window,, tumor suppression might critically dependd on the presence of Rb as the sole safeguard.. Therefore, the identification of the complementingg mutations required in mice to causee retinoblastoma might also point to pathwayss that are deficient in the human retina.. If this is indeed the case, one then wonderss whether this is a sheer accident of evolutionn or the consequence of a selection processs that warrants further investigation.

Methods s

GenerationGeneration of mice Usingg homologous recombination in embryonic stemm (ES) cells two 34 bp lox? sites (5'-ATAACTTCGTATAATGTATGCTATACGAA A GTTAT-3')) with the complementary loxP site wheree introduced into the mouse Rb gene flankingg exon 19 in the identical position as describedd previously for RbF*T{9 mice (Vooijs et al.. 1998). Briefly, /ƒƒ>/? F-deficient HM-\ ES cells weree electroporated with the RbloxP targeting vectorr and selected for one week in the presence off HAT containing medium (Gibco-BRL). Homologouss recombinants (92 %) were expanded,, karyotyped and reseeded at low densityy (lxl05/60 cm2) in conditioned medium containingg 10 uM 6-ThioGuanine (6-TG, Sigma).. Spontaneous revertants were subcloned, expanded,, karyotyped and injected into recipient blastocysts.. Homologous recombinants were identifiedd by Southern blot analysis as described previouslyy (Vooijs et al. 1998). Mice with targetedd null alleles for Trp53 and pi07 have beenn described (Donehower et al. 1992; Robanus-Maandagg et al. 1998). Photoreceptor-specific Cree expression was achieved using the human Interphotoreceptorr Retinol Binding Protein (IRBP)) promoter. A 1.3 kb fragment containing thee hIRBP promoter (A generous gift from G. Liou)) was cloned into a vector containing a nuclear-localizedd Cre (nls Cre) and rabbit p*-globinn splicing and polyadenylation signals,

isolatedd by Notl-Sall digestion and prepared for microinjectionn into fertilized FVB/N oocytes. Twoo different transgenic lines where characterizedd designated IC-47 and IC-64 that aree used in this study (Akagi et al. 1997). Deletionn of RbF'9 alleles in the germ line was achievedd by crossing with the Actin-cre deleter strainn (M. Vooijs and A. Berns, unpublished results). .

ProteinProtein analysis Primaryy keratinocytes were isolated from newbornn mice and cultured as described (Vooijs et al.. 2001). Protein lysates were made directly fromm plates of growing keratinocytes using ice-coldd RIPA buffer. Total cell extracts were sequentiallyy immunoprecipitated with antibodies againstt Rb, pi07 and pi30 and separated on 5% PAGE,, transferred to PDVF membrane (Millipore)) followed by western blot analysis withh the same antibodies except for Rb, a mouse monoclonall G3-245 (PharMingen) was used was usedd for western blot analysis. Antibodies used weree Rb(C-15), pl07 (C-18), pl30 (C-20) and weree obtained from Santa Cruz. Goat anti-rabbit andd goat anti-mouse secondary antibodies were fromm Biosource.

HistologicalHistological and immunohisotchemical analysis. Forr routine histology tissues were fixed in 4% bufferedd formalin. Sections of 5 u.m were deparaffinizedd and stained with haematoxilin-eosinn (HE). Whole mount p"-galactosidasee staining was performed on freshly isolatedd tissues as described (Akagi et al. 1997). Forr indirect immunohistochemistry using DAB substrate,, sections were blocked in methanol/3% H2022 to remove endogenous peroxidase activity andd blocked in PBS containing 10% normal goat serum,, 1% BSA. Primary antibodies were incubatedd o/n at 4 °C in PBS, 1% BSA. Polyclonall antisera were obtained from Harlan (a-MSH,, ACTH, p-endorphin, LH/FSH or from DAKOO (BrdU, Prolactin, Growth Hormone, Synaptophysinn and GFAP). Rabbit antisera againstt rabbit-p"-TSH (AFP-1274789) and rat-a-glycoproteinn subunit (AFP-66P9986), human-S-antigenn and monkey-lRBP were gifts. Immunostainingg using a polyclonal antiserum againstt Cre (1:15000, Novagen) required antigen retrievall in 10 mM citrate-buffer. Biotinylated

61 1

ChapterChapter 4

secondaryy antibodies (Biosource) were detected usingg the ABC system (DAKO). Tunel labeling waswas performed according to the manufacturer on proteinase-KK treated paraffin-embedded sections (ITK,, Diagnostics). For the analysis of BrdU incorporation,, mice were injected i.p. with 100 ug/grr bodyweight BrdU (PBS, 7 mM NaOH) and sacrificedd 2-3 hrs later.

TissueTissue DNA analysis and genotyping DNAA from ES cells or tissues was isolated as describedd (Laird et al. 1991). Genotyping of mice waswas performed using PCR on tail tip DNA. PI07 knockoutt mice were analyzed with a 4-primerset: LZ11 (5'CGTCACACTACGTCTGA-ACG-3') and LZ22 (5'-CGACCAGATGATCA-CACTCG-3') for thee mutant allele (560 bp) and pl07F2 (5TCGTGAGCGCGGATAGAAAGG-3')) and pl07RR (5'-CTGCCGAAGGTAAATACGGAC-3') forr the wild type fragment (216 bp). Rb floxed micee were analyzed using a primer-set Rbl8 (5'-GGCGTGTGCCATCAATG-3')) and Rbl9 (5'-AACTCAAGGGAGACCTG-3')) yielding products off 678 bp (Rb-wt), 746 (Rb-F19) and 314 bp (Rb-A19),, respectively. TRP53 KO mice were analyzedd as described yielding 430 bp (wt) and 3900 bp (ko) products (Marino et al. 2000). PCR reactionn and cycling conditions were similar for alll genotypes except for Rb where 9% glycerol andd MgCl2 was 1.8 mM (Vooijs et al. 1998). AcZLAcZL reporter mice were analyzed as described (Akagii et al. 1997). The rd status of mice was determinedd as described (Pittler et al. 1991). Transgenicc hIRBP-cre founders were identified byy EcoRV digestion of tail DNA and Southern blott analysis, using a Cre probe. Established lines weree further analyzed by PCR using Crel (5'-GCACGTTCACCGGCATCAAC-3')) and Cre2 (5'-CGATGCAACGAGTGATGAGGTTC-3')) yielding aa 270-bp product. Micro dissection of the ONL off the retina was performed using a laser capture micro-dissectionn (Arcturus) on 10 (xm HE-stainedd formalin-fixed deparaffinzed sections thatt were extensively dehydrated in xylene and dried.. The dissected material was transferred to 500 uJ of proteinase-K buffer and incubated at 37°CC o/n, after which the proteinase-K was inactivatedd for 10 min at 96°C followed by 35 cycless of PCR after a 'hot start' with Hot-wax MgCl22 (1.5 mM) beads (Invitrogen) as described (Vooijss et al. 1998).

Acknowledgements s

Wee wish to thank B. Wiggert (National Eye Institute,, NIH, Bethesda), T. Shinohara (BWH, Harvard,, Boston) and A. Parlow (NIDKD, NIH, Bethesda)) for antibodies and G. Liou for the IRBPP promoter fragment (Medical College of Georgia,, Augusta). J. Zevenhoven, D. Hoogervorst,, M. Tjin-a-Koeng, K. de Goeij and J.. Bulthuis for histo-technical assistance (Histology-coree facility, NKI Amsterdam); N. Bosnië,, F. van der Ahe, A. Zwerver, T. Maidmentt and S. Greven for animal husbandry andd E. Danen for help with protein analysis. We thankk Erik Danen, S. Ruiz, E. Robanus-Maandag andd K. Quon for useful comments on the manuscriptt and Jos Jonkers for providing the tumorr incidence curve of Trp53+/~ mice. This workk was supported through a program grant fromm the Netherlands Organization of Scientific Researchh (M.V.) and a grant from the Dutch Cancerr Society (NKB/KWF to A.B. and M.V.)

References s

Akagi,, K., Sandig, V., Vooijs, M., van der Valk, M.,, Giovannini, M., Strauss, M , and Berns,, A. 1997. Cre-mediated somatic site-specificc recombination in mice. Nucleic AcidsAcids Research 25: 1766-1773.

al-Ubaidi,, M.R., Font, R.L., Quiambao, A.B., Keener,, M.J., Liou, G.I., Overbeek, P.A., andd Baehr, W. 1992. Bilateral retinal and brainn tumors in transgenic mice expressing simiann virus 40 large T antigen under controll of the human interphotoreceptor retinoid-bindingg protein promoter. J Cell 5/0/119:: 1681-1687.

Bader,, J.L., Meadows, A.T., Zimmerman, L.E.,, Rorke, L.B., Voute, P.A., Champion, L.A.,, and Miller , R.W. 1982. Bilateral retinoblastomaa with ectopic intracranial retinoblastoma:: trilateral retinoblastoma. CancerCancer Genet Cytogenet 5: 203-213.

Blackshaw,, S. and Snyder, S.H. 1997. Developmentall expression pattern of phototransductionn components in mammaliann pineal implies a light-sensing function.. JNeurosci 17: 8074-8082.

Bogenmann,, E., Lochrie, M.A., and Simon, M.I .. 1988. Cone cell-specific genes

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TumorTumor formation in mice with somatic Rb loss

expressedd in retinoblastoma. Science 240: 76-78. .

Bowes,, C, Li, T., Danciger, M., Baxter, L.C., Applebury,, M.L., and Farber, D.B. 1990. Retinall degeneration in the rd mouse is causedd by a defect in the beta subunit of rod cGMP-phosphodiesterase.. Nature 347: 677-680. .

Carter-Dawson,, L.D. and LaVail, M.M. 1979. Rodss and cones in the mouse retina. II. Autoradiographicc analysis of cell generation usingg tritiated thymidine. J Comp Neurol 188:263-272. .

Carter-Dawson,, L.D., LaVail, M.M., and Sidman,, R.L. 1978. Differential effect of the rdd mutation on rods and cones in the mouse retina.. Invest Ophthalmol Vis Sci 17: 489-498. .

Cepko,, C.L., Austin, C.P., Yang, X., Alexiades,, M., and Ezzeddine, D. 1996. Celll fate determination in the vertebrate retina.. Proc Natl Acad Sci USA 93: 589-595.

Chen,, P.L., Riley, D.J., Chen, Y., and Lee, W.H.. 1996. Retinoblastoma protein positivelyy regulates terminal adipocyte differentiationn through direct interaction withh C/EBPs. Genes and Development 10: 2794-2804. .

Clarke,, A.R., Robanus-Maandag, E., van Roon, M.,, van der Lugt, N.M., van der Valk, M., Hooper,, M.L., Berns, A., and te Riele, H. 1992.. Requirement for a functional Rb-1 genee in murine development. Nature 359: 328-330. .

Claudio,, P.P., Howard, CM., Pacilio, C, Cinti,, C, Romano, G., Minimo, C, Maraldi,, N.M., Minna, J.D., Gelbert, L., Leoncini,, L. et al. 2000. Mutations in the retinoblastoma-relatedd gene RB2/pl30 in lungg rumors and suppression of tumor growthh in vivo by retrovirus-mediated gene transfer.. Cancer Res 60: 372-382.

Donehower,, L.A., Harvey, M., Slagle, B.L., McArthur,, M.J., Montgomery, C.A., Butel,, J.S., and Bradley, A. 1992. Mice deficientt for p53 are developmentally normall but susceptible to spontaneous tumours.. Nature 356:215-221.

Dyson,, N. 1998. The regulation of E2F by pRB-familyy proteins. Genes and DevelopmentDevelopment 12: 2245-2262.

Friend,, S.H., Bernards, R., Rogelj, S., Weinberg,, R.A., Rapaport, J.M., Albert, D.M.,, and Dryja, T.P. 1986. A human DNAA segment with properties of the gene thatt predisposes to retinoblastoma and osteosarcoma.. Nature 323: 643-646.

Fung,, Y.K., Murphree, A.L., T'Ang, A., Qian, J.,, Hinrichs, S.H., and Benedict, W.F. 1987. Structurall evidence for the authenticity of thee human retinoblastoma gene. Science 236:: 1657-1661.

Gallie,, B.L., Campbell, C, Devlin, H., Duckett,, A., and Squire, J. A. 1999. Developmentall basis of retinal-specific inductionn of cancer by RB mutation. CancerCancer Res 59: 1731-1735.

Geng,, Y., Whoriskey, W., Park, M.Y., Bronson,, R.T., Medema, R.H., Li, T., Weinberg,, R.A., and Sicinski, P. 1999. Rescuee of cyclin D1 deficiency by knockin cyclinE.Ce//97:: 767-777.

Geng,, Y., Yu, Q., Sicinska, E., Das, M., Bronson,, R.T., and Sicinski, P. 2001. Deletionn of the p27Kipl gene restores normall development in cyclin Dl-deficient mice.. Proc Natl Acad Sci USA 98: 194-199.

Gonzalez-Fernandez,, F., Lopes, M.B., Garcia-Fernandez,, J.M., Foster, R.G., De Grip, W.J.,, Rosemberg, S., Newman, S.A., and VandenBerg,, S.R. 1992. Expression of developmentallyy defined retinal phenotypes inn the histogenesis of retinoblastoma. Am J PatholPathol 141: 363-375.

Griep,, A.E., Krawcek, J., Lee, D., Liem, A., Albert,, D.M., Carabeo, R., Drinkwater, N., McCall,, M., Sattler, C, Lasudry, J.G. et al. 1998.. Multiple genetic loci modify risk for retinoblastomaa in transgenic mice. Invest.Ophthalmol.Vis.Sci.. 39: 2723-2732.

Hanahan,, D. and Weinberg, R.A. 2000. The hallmarkss of cancer. Cell 100: 57-70.

Harbour,, J.W., Lai, S.L., Whang-Peng, J., Gazdar,, A.F., Minna, J.D., and Kaye, F.J. 1988.. Abnormalities in structure and expressionn of the human retinoblastoma genee in SCLC. Science 241: 353-357.

Harbour,, J.W., Luo, R.X., Dei Santi, A., Postigo,, A.A., and Dean, D.C. 1999. Cdk phosphorylationn triggers sequential intramolecularr interactions that progressivelyy block Rb functions as cells

63 3

ChapterChapter A

movee through Gl. Cell 98: 859-869. Howes,, K.A., Lasudry, J.G., Albert, D.M.,

andd Windle, J.J. 1994a. Photoreceptor cell tumorss in transgenic mice. Invest OphthalmolOphthalmol Vis Sci 35: 342-351.

Howes,, K.A., Ransom, N., Papermaster, D.S., Lasudry,, J.G., Albert, D.M., and Windle, J.J.. 1994b. Apoptosis or retinoblastoma: alternativee fates of photoreceptors expressingg the HPV-16 E7 gene in the presencee or absence of p53. Genes and Development^:Development^: 1300-1310.

Hu,, N., Gutsmann, A., Herbert, D.C., Bradley, A.,, Lee, W.H., and Lee, E.Y. 1994. Heterozygouss Rb-1 delta 20/+mice are predisposedd to tumors of the pituitary glandd with a nearly complete penetrance. Oncogene^.Oncogene^. 1021-1027.

Jacks,, T., Fazeli, A., Schmitt, E.M., Bronson, R.T.,, Goodell, M.A., and Weinberg, R.A. 1992.. Effects of an Rb mutation in the mouse.. Nature 359: 295-300.

Jakobiec,, F.A., Tso, M.O., Zimmerman, L.E., andd Danis, P. 1977. Retinoblastoma and intracraniall malignancy. Cancer 39: 2048-2058. .

Knudson,, A.G., Jr. 1971. Mutation and cancer: statisticall study of retinoblastoma. Proc NatlNatl Acad Sci USA 68: 820-823.

Laird,, P.W., Zijderveld, A., Linders, K., Rudnicki,, M.A., Jaenisch, R., and Berns, A. 1991.. Simplified mammalian dna isolation procedure.. Nucleic Acids Research 19: 4293. .

Lasorella,, A., Noseda, M , Beyna, M., and Iavarone,, A. 2000. Id2 is a retinoblastoma proteinn target and mediates signalling by Mycc oncoproteins. Nature 407: 592-598.

Lee,, E.Y., To, H., Shew, J.Y., Bookstein, R., Scully,, P., and Lee, W.H. 1988. Inactivation off the retinoblastoma susceptibility gene in humann breast cancers. Science 241: 218-221. .

Lee,, E.Y., Chang, C.Y., Hu, N., Wang, Y.C., Lai,, C.C., Herrup, K., Lee, W.H., and Bradley,, A. 1992. Mice deficient for Rb are nonviablee and show defects in neurogenesis andd haematopoiesis. Nature 359: 288-294.

Lee,, M.H., Williams, B.O., Mulligan, G., Mukai,, S., Bronson, R.T., Dyson, N., Harlow,, E., and Jacks, T. 1996.

Targetedd disruption of pi07: functional overlapp between pi07 and Rb. Genes and DevelopmentDevelopment 10: 1621-1632.

Lee,, W.H., Bookstein, R., Hong, F., Young, L.J.,, Shew, J.Y., and Lee, E.Y. 1987. Humann retinoblastoma susceptibility gene: cloning,, identification, and sequence. ScienceScience 235: 1394-1399.

Levy,, A., Hall, L., Yeudall, W.A., and Lightman,, S.L. 1994. p53 gene mutations in pituitaryy adenomas: rare events. Clin EndocrinolEndocrinol 41: 809-814.

Liou,, G.I., Geng, L., al, U.M., Matragoon, S., Hanten,, G., Baehr, W., and Overbeek, P.A. 1990.. Tissue-specific expression in transgenicc mice directed by the 5'- flanking sequencess of the human gene encoding interphotoreceptorr retinoid-binding protein. JJ Biol Chem 265: 8373-8376.

Liou,, G.I., Wang, M., and Matragoon, S. 1994. Timingg of interphotoreceptor retinoid-bindingg protein (IRBP) gene expression and hypomethylationn in developing mouse retina.. Dev Biol 161: 345-356.

Margo,, C, Hidayat, A., Kopelman, J., and Zimmerman,, L.E. 1983. Retinocytoma. A benignn variant of retinoblastoma. Arch OphthalmolOphthalmol 101: 1519-1531.

Marino,, S., Vooijs, M., van Der Gulden, H., Jonkers,, J., and Berns, A. 2000. Induction off medulloblastomas in p53-null mutant micee by somatic inactivation of Rb in the externall granular layer cells of the cerebellum.. Genes and Development 14: 994-1004. .

Martinez,, L.A., Chen, Y., Fischer, S.M., and Conti,, C.J. 1999. Coordinated changes in celll cycle machinery occur during keratinocytee terminal differentiation. OncogeneOncogene 18: 397-406.

Metzger,, D. and Feil, R. 1999. Engineering the mousee genome by site-specific recombination.. Curr Opin Biotechnol 10: 470-476. .

Mihara,, K., Cao, X.R., Yen, A., Chandler, S., Driscoll,, B., Murphree, A.L., T'Ang, A., andd Fung, Y.K. 1989. Cell cycle-dependent regulationn of phosphorylation of the human retinoblastomaa gene product. Science 246: 1300-1303. .

64 4

TumorTumor formation in mice with somatic Rb loss

Moll ,, A.C., Imhof, S.M., Bouter, L.M., and Tan,, K.E. 1997. Second primary tumors in patientss with retinoblastoma: A review of thee literature. Opthalmic Genet 18(1): 27-34. .

Mulligan,, G. and Jacks, T. 1998. The retinoblastomaa gene family: cousins with overlappingg interests. Trends in Genetics 14:: 223-229.

Nikitin,, A.Y., Juarez-Perez, M.I., Li, S., Huang,, L., and Lee, W.H. 1999. RB-mediatedd suppression of spontaneous multiplee neuroendocrine neoplasia and lung metastasess in Rb+/- mice. Proc Natl Acad SciUSASciUSA 96: 3916-3921.

Nork,, T.M., Schwartz, T.L., Doshi, H.M., and Millecchia,, L.L. 1995. Retinoblastoma. Cell off origin. Arch.Ophthalmol. 113: 791-802.

Pittler,, S.J. and Baehr, W. 1991. Identification off a nonsense mutation in the rod photoreceptorr cGMP phosphodiesterase beta-subunitt gene of the rd mouse. Proc NatlNatl Acad Sci USA 88: 8322-8326.

Riley,, D.J., Nikitin, A.Y., and Lee, W.H. 1996. Adenovirus-mediatedd retinoblastoma gene therapyy suppresses spontaneous pituitary melanotrophh tumors in Rb+/- mice. Nature MedicineMedicine 2: 1316-1321.

Robanus-Maandag,, E., Dekker, M., van der Valk,, M., Carrozza, M.L., Jeanny, J.C., Dannenberg,, J.H., Berns, A., and te Riele, H.. 1998. pi 07 is a suppressor of retinoblastomaa development in pRb-deficientt mice. Genes and Development 12: 1599-1609. .

Robanus-Maandag,, E., van der Valk, M., Vlaar, M.,, Feltkamp, C, O'Brien, J., van Roon, M.,, van der Lugt, N., Berns, A., and te, R. 1994.. Developmental rescue of an embryonic-lethall mutation in the retinoblastomaa gene in chimeric mice. Embo 13:4260-4268. .

Sherr,, C.J. and Roberts, J.M. 1999. CDK inhibitors:: positive and negative regulators off Gl-phase progression. Genes and Development^:Development^: 1501-1512.

Sicinski,, P., Donaher, J.L., Parker, S.B., Li, T., Fazeli,, A., Gardner, H., Haslam, S.Z., Bronson,, R.T., Elledge, S.J., and Weinberg, R.A.. 1995. Cyclin Dl provides a link betweenn development and oncogenesis in thee retina and breast. Cell 82: 621 -630.

Slack,, R.S., El-Bizri, H., Wong, J., Belliveau, D.J.,, and Miller, F.D. 1998. A critical temporall requirement for the retinoblastoma proteinn family during neuronal determination.. J Cell Biol 140: 1497-1509.

Takahashi,, R., Hashimoto, T., Xu, H.J., Hu, S.X.,, Matsui, T., Miki , T., Bigo-Marshall, H.,, Aaronson, S.A., and Benedict, W.F. 1991.. The retinoblastoma gene functions as aa growth and tumor suppressor in human bladderr carcinoma cells. Proc Natl Acad Sci USAUSA 88: 5257-5261.

Tsumanuma,, I., Sato, M., Okazaki, H., Tanaka,, R., Washiyama, K.., Kawasaki, T., andd Kumanishi, T. 1995. The analysis of p533 tumor suppressor gene in pineal parenchymall tumors. Noshuyo Byori 12: 39-43. .

Vooijs,, M., van der Valk, M., te Riele, H., and Berns,, A. 1998. Flp-mediated tissue-specificc tnactivation of the retinoblastoma tumorr suppressor gene in the mouse. OncogeneOncogene 17: 1-12.

Vooijs,, M. and Berns, A. 1999. Developmental defectss and tumor predisposition in Rb mutantt mice. Oncogene 18: 5293-5303.

Vooijs,, M., Jonkers, J., and Berns, A. 2001. A highlyy efficiënt ligand-regulated Cre recombinasee mouse line shows that LoxP recombinationn is position dependent. EMBOEMBO Rep 2: 292-297.

Weinberg,, R.A. 1995. The retinoblastoma proteinn and cell cycle control. Cell 81: 323-330. .

Williams,, B.O., Remington, L., Albert, D.M., Mukai,, S., Bronson, R.T., and Jacks, T. 1994a.. Cooperative tumorigenic effects of germlinee mutations in Rb and p53. Nature GeneticsGenetics 7: 480-484.

Williams,, B.O., Schmitt, E.M., Remington, L., Bronson,, R.T., Albert, D.M., Weinberg, R.A.,, and Jacks, T. 1994b. Extensive contributionn of Rb-defictent cells to adult chimericc mice with limited histopathological consequences.. Embo 13: 4251-4259.

Williams,, R.W. and Goldowitz, D. 1992. Structuree of clonal and polyclonal cell arrayss in chimeric mouse retina. Proc Natl AcadAcad Sci USA 89: 1184-1188.

65 5

ChapterChapter A

Woloschak,, M., Roberts, J.L., and Post, K.D. 1994.. Loss of heterozygosity at the retinoblastomaa locus in human pituitary tumors.. Cancer 74: 693-696.

Woloschak,, M , Yu, A., and Post, K.D. 1997. Frequentt inactivation of the pl6 gene in humann pituitary tumors by gene methylation.. Mol Mol Carcinog 19: 221-224.

Yokoyama,, T., Liou, G.I., Caldwell, R.B., and Overbeek,, P.A. 1992. Photoreceptor-specificc activity of the human interphotoreceptorr retinoid-binding protein (IRBP)) promoter in transgenic mice. Exp EyeEye Res 55: 225-233.

Young,, R.W. 1984. Cell death during differentiationn of the retina in the mouse. J CompComp Neurol 229: 362-373.

-.. 1985a. Cell differentiation in the retina of the mouse.. AnatRec 212: 199-205.

-.. 1985b. Cell proliferation during postnatal developmentt of the retina in the mouse. BrainBrain Res 353: 229-239.

Zhang,, H.S., Postigo, A.A., and Dean, D.C. 1999.. Active transcriptional repression by thee Rb-E2F complex mediates Gl arrest triggeredd by pl6INK4a, TGFbeta, and contactt inhibition. Cell 97: 53-61.

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