Received: 13 March 2002Accepted: 30 July 2002Published online: 28 September 2002© Springer-Verlag 2002

Abstract Background and aims:Recently we demonstrated that phos-phatidylinositol 3-kinase (PI3K) isoverexpressed in human lung cancer.This study evaluated whether thePI3K inhibiting agent wortmanninaffects proliferation of human lungcancer cells in vitro and in vivo.Methods: Effects of exposure of hu-man non-small-cell lung cancer(NSCLC) cells (KNS-62, Colo-699)to wortmannin were investigated invitro by proliferation, cytotoxicity,and DNA fragmentation assays. Invivo we examined the effects ofblocking PI3K by wortmannin priorto xenotransplantation of humanNSCLC cells into SCID-bg mice andthe effect of systemic wortmanninadministration following intrapulmo-nary xenotransplantation of humanNSCLC. Results: Exposure of KNS-62 and Colo-699 lung cancer cells to

wortmannin inhibited proliferation incorrelation to concentration in vitro.In vivo the blocking of PI3K bywortmannin prior to xenotransplan-tation caused a significant delay inthe growth of subcutaneously in-duced tumors. Systemic wortmanninadministration increased mean sur-vival after intrapulmonary xeno-transplantation of human NSCLCsignificantly by 38% and 47%. Con-clusions: These data suggest inhibi-tion of PI3K activity as a potentialtarget for treatment of humanNSCLC. Systemic toxicity ofwortmannin requires development ofimproved PI3K inhibitors with fa-vorable pharmacological properties.

Keywords Human lung cancer · Intrapulmonary xenotransplantationmodel · Phosphatidylinositol 3-kinase · Wortmannin

Langenbeck’s Arch Surg (2002) 387:234–239DOI 10.1007/s00423-002-0314-x O R I G I N A L A RT I C L E

Arnd Steffen BoehleRoland KurdowLars BoenickeBodo SchniewindFred FaendrichPeter DohrmannHolger Kalthoff

Wortmannin inhibits growth of human non-small-cell lung cancer in vitro and in vivo

Introduction

We recently demonstrated that phosphatidylinositol 3-ki-nase (PI3K) is overexpressed in non-small-cell humanlung cancer (NSCLC) [1]. PI3K is a heterodimer com-posed of a p85 regulatory subunit and a p110 catalyticsubunit [2]. The products of PI3K function as secondmessenger molecules for regulation of cell growth, pro-liferation, and apoptosis in response to various growthhormones [3]. PI3K is required for oncogenic tyrosinekinases/receptors to induce tumor cell growth and prolif-eration [4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. The p85 and p110subunits of PI3K are overexpressed at protein level inprimary lung carcinomas irrespective of the histological

type, and PI3K overexpression is correlated with tumorgrading [1]. In contrast, no overexpression is observed innormal lung tissue or benign lesions. PI3K has beenidentified as a potential target for anticancer drug devel-opment because of its role as a component of growth fac-tor and oncogene activated signaling pathways [14].Wortmannin (C23H2408), a highly cell permeable fungalmetabolite from Penicillium fumiculosum, acts as a se-lective, irreversible inhibitor of the catalytic p110 PI3Ksubunit [15, 16, 17, 18].

As PI3K is overexpressed in NSCLC, the aim of thisstudy was to evaluate whether selective inhibition of thePI3K activity by its inhibitor wortmannin affects prolif-eration of human lung cancer cells in vitro and in vivo.

A.S. Boehle (✉) · R. KurdowL. Boenicke · B. Schniewind · F. FaendrichP. Dohrmann · H. KalthoffDepartment for General Surgery and Thoracic Surgery, Christian Albrecht University, Arnold-Heller-Strasse 7, 24105 Kiel, Germanye-mail: boehle@surgery.uni-kiel.deTel.: +49-4102-5974305Fax: +49-4102-5974586

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Material and methods

Cell lines and culture conditions

Human lung cancer cells KNS-62 [19] derived from the brain me-tastasis of a squamous cell carcinoma of the lung and Colo-699 [20]derived from adenocarcinoma of the lung were cultured in RPMI1640 supplemented with 10% fetal calf serum, 1% sodium pyru-vate, and 1% L-glutamine (Life Technologies, Paisley, UK). Cellsgrew as monolayers in 75 cm2 flasks at 37°C in a humidified incu-bator gassed with 5% CO2 and 95% air. When monolayers grew toapproximately 80% confluence, cells were subcultured or harvestedusing trypsin EDTA (Life Technologies). Cells to be xenotrans-planted were equilibrated in serum-free culture medium at a densityof 2×106 cells per 50 µl injection volume. Cell viability was testedby trypan blue staining. All cell cultures were confirmed to be freeof Mycoplasma infection by reverse transcriptase polymerase chainreaction of supernatants from densely growing cells following theinstructions of the manufacturer (Takara Shuzo, Japan).

[3H]Thymidine incorporation assay

Tumor cells (1×104; KNS-62, Colo-699) were seeded in 100 µlculture medium in 96-well microtiter plates. Cells were exposed towortmannin (Sigma-Aldrich, Munich, Germany) in concentrationsranging from 10 to 400 nM for 24 or 48 h. To each well we added0.02 µCi tritiated thymidine (43.0 Ci/mmol, Amersham PharmaciaBiotech, Braunschweig, Germany) 3 h before cell harvest. Afterwashing with phosphate-buffered solution (PBS) three times cellswere lysed by incubation with trypsin EDTA. The lysate was fil-trated through a glass fiber filtermat (Wallac Oy, Turku, Finland)by a 96-well cell harvester (Inotech). The filtermat was placed in aMicroBeta sample bag together with 4.5 ml scintillation fluid.Counting was performed in a 1450 MicroBeta scintillation counter(Wallac Oy)

The relative recovery of incorporated [3H]thymidine in theDNA of proliferating cells was estimated for determination of in-hibition of proliferation. Each measurement was performed sixtimes.

Cell viability assay

To determine wortmannin-induced cytotoxicity cell viability assayswere performed following the manufacturers instructions (EZ4U,Biomedica, Vienna, Austria). Tumor cells (5×103; KNS-62, Colo-699) in 200 µl culture medium per well were seeded in 96-wellmicrotiter plates. Cells were exposed to CA-4PD in concentrationsranging from 10–8 to 10–4 M for 3 or 24 h. Cell viability was esti-mated photometrically. Each measurement was performed sixtimes.

DNA fragmentation assay (JAM assay)

The DNA fragmentation test was performed as originally de-scribed [21]. In 96-well microtiter plates we seeded 1×104 cells in100 µl culture medium per well. Cells were incubated for 3 h with0.05 µCi tritiated thymidine. Medium was removed, and cells werewashed twice in PBS. Cells were then incubated with wortmanninin concentrations ranging from 10 to 400 nM for 24 or 48 h. Ad-herent and floating cells were lysed by 100 µl 0.2% sodium dode-cyl sulfate buffer. The lysate was processed as described above.The proportion of initially incorporated label was quantified. Asfragmented DNA of apoptotic cells can pass the filtermat, the pro-portion of apoptotic cells can be estimated by the loss of initiallyincorporated label. Each sample was prepared six times.

Experimental animals

Pathogen-free female SCID-bg mice (Harlan Winkelmann, Bor-chen, Germany) [22] were maintained in sterile polycarbonate mi-croisolator cages under pathogen-free conditions, fed autoclavedfood and water ad libidum, and handled under stringent sterileconditions in a laminar flow hood.

To test whether inhibition of PI3K activity affects tumor cellproliferation in vivo tumor cells were incubated with wortmanninfor 6 h prior to xenotransplantation at a concentration of 100 nM.Tumor cells were isolated, washed in PBS, and adjusted to 2×106

tumor cells per 50 µl serum-free culture medium. For the induc-tion of subcutaneous tumor growth 2×106 tumor cells (KNS-62,Colo-699) were injected dorsally into the subcutis of the left flank.Animals were randomly distributed into groups of seven animalseach and received tumor cells either pretreated by wortmannin orby PBS alone, which served as control.

Animals were monitored daily for local tumor growth, and tu-mors were gauged percutaneously in two diameters. Tumor vol-ume was calculated by: V=L×W2×0.52 (V is volume, L is length,W is width) [23]. Animals were killed 28 days after subcutaneousxenotransplantation by CO2 inhalation and tumors removed forhistological examination.

An orthotopic xenotransplantation model was used to deter-mine the systemic effect of wortmannin administration on thegrowth of human lung cancer in vivo. For induction of intrapul-monary tumor growth mice were anesthetized by intraperitonealinjection of 240 mg/kg Avertin [24], and 2×106 tumor cells wereintroduced below the visceral pleura as described previously [20].

Animals were randomly distributed into treatment groups andcontrol groups of seven animals each. Treatment was started onday 2 postoperatively by daily systemic administration of 1 mg/kgwortmannin intraperitoneally. Animals serving as controls wereinjected by sterile PBS in corresponding volumes. All animalswere monitored daily for signs of respiratory distress or physicaldiscomfort. When animals became respiratory insufficient, theywere killed by CO2 inhalation, and tumors were removed for his-tological examination.

All animal experiments were approved by the Review Board ofthe Ministry of Schleswig-Holstein, and the guidelines of Interdis-ciplinary Principles and Guidelines for the Use of Animals in Re-search, Testing and Education issued by the New York Academyof Sciences were observed strictly.

Histological examination

Tissues were fixed in formalin and stained with hematoxyllinand eosin using standard procedures. For immunohistochemistryfresh tumor tissue was snap frozen in liquid nitrogen. Antibodiesto PI3K p85 (Z-8) and p110 (C-17) were obtained from SantaCruz Biotechnology (Heidelberg, FRG). Staining and immuno-histochemical scoring was performed as published previously[1].

Immunocompetence assay

When animals were killed, blood was withdrawn from the abdom-inal vein, allowed to clot and serum was isolated by centrifuge.Cryopreserved histological sections of the corresponding tumorwere fixated by acetone, air dried and incubated at 4°C overnightwith 50 µl murine serum. Sections were washed in PBS for threetimes and incubated for 1 h with a peroxidase coupled rabbit anti-mouse IgG and IgM antibody in 1:1000 dilution (Dianova, Ham-burg, Germany). After careful rinsing by PBS staining was per-formed adding peroxidase H2O2 diaminobenzidine substrate solu-tion for 10 min. None of the animals had gained significant immu-nocompetence during the in vivo studies.

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Statistical analysis

Descriptive statistics and Student’s t test calculated by Primer ofBiostatistics 3.01 (RAmEx, Los Angeles, Calif., USA).

Results

[3H]Thymidine incorporation assay

Proliferating lung cancer cells were exposed towortmannin in different concentrations for various incu-bation periods in a [3H]thymidine incorporation assay forthe determination of a potentially cytostatic effect. Expo-sure of proliferating KNS-62 cells to wortmannin inhib-ited cell proliferation in correlation to drug concentra-tion. After a 24-h incubation period a concentration of50 nM reduced tumor cell proliferation by 19% com-pared to untreated controls. A concentration of 400 nMwortmannin induced an almost complete stop of tumorcell proliferation. Prolongation of drug exposure periodto 48 h did not induce a significant increase in prolifera-tion inhibition (Fig. 1A).

Exposure of proliferating Colo-699 cells displayed asimilar pattern of inhibition of tumor cell proliferation incorrelation to drug concentration. A concentration of50 nM was efficient to reduce tumor cell proliferation by17%; a concentration of 400 nM induced an almost com-plete stop of tumor cell proliferation. Increase in the pe-riod of drug exposure to 48 h did not increase the inhibi-tory effect (Fig. 1B).

Cell viability assay

Cell viability assays were performed for determinationof wortmannin induced cytotoxicity. Corresponding to[3H]thymidine assays KNS-62 and Colo-699 lung cancercells were exposed to wortmannin in concentrationsranging from 10 to 400 nM. There was no statisticallysignificant cytotoxicity induced by wortmannin exposurein vitro for 24 or 48 h.

DNA fragmentation assay (JAM assay)

Determination of fragmented DNA indicative of apopto-sis following tumor cell exposure to wortmannin demon-strated a slight increase in the proportion of apoptotic tu-mor cells from 6% in controls to 11% following drug ex-posure at 400 nM (n.s.).

Histological examination

Subcutaneously induced tumors grew as solitary lesionswithout local or systemic metastases. Histopathological

examination confirmed for KNS-62 the growth of a solidsquamous lung carcinoma with a G2 differentiation. Thetumors appeared to have a relative sharp border and dis-played moderate invasion into the surrounding tissue.Colo-699 injection induced huge tumors reflecting apoorly differentiated (G3) adenocarcinoma of the lung.In comparison to KNS-62 these tumors grew locallymore aggressively, infiltrating the surrounding tissue.

Both tumors induced by subcutaneous xenotransplan-tation of either KNS-62 or Colo-699 lung cancer cellsstrongly overexpressed p85 and p110 subunits of PI3Kimmunohistochemically.

Xenotransplantation of PI3K blocked tumor cells

Selective blocking of PI3K activity by incubation withwortmannin at a concentration of 100 nM for 6 h prior to

Fig. 1 A Exposure of proliferating KNS-62 lung cancer cells towortmannin resulted in a dose-dependent inhibition of tumor cellproliferation. A dose of 200 nM induced a highly significant inhi-bition of proliferation 24 h after drug exposure compared to un-treated controls; **P<00.1, error bars SD. Prolongation of drugexposure to 48 h slightly increased the cytostatic effect in vitro. B Exposure of proliferating Colo-699 lung cancer cells towortmannin resulted in a comparable inhibition of dose-dependenttumor cell proliferation. A dose of 200 nM was required to inducea highly significant inhibition of proliferation compared to un-treated controls, while prolongation of drug exposure to 48 h re-duced this to 100 nM; **P<00.1, error bars SD

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xenotransplantation caused a significant delay in thegrowth of subcutaneously induced tumors in comparisonto untreated references. These differences were statisti-cally significant on day 21 following tumor cell inocula-tion (P<0.05). On day 28 after xenotransplantation insquamous cell carcinoma (KNS-62) mean tumor volumewas 135±91 mm3 after wortmannin pretreatment, com-pared to 508±376 mm3 in the reference group (Fig. 2A).In adenocarcinoma (Colo-699) mean tumor volume onday 28 after PI3K blocking by wortmannin was117±94 mm3, compared to 523±232 mm3 in the refer-ence group (Fig. 2B.) There was no systemic toxicity ob-served in animals of the wortmannin group.

Systemic administration of wortmannin in intrapulmonaryxenotransplanted NSCLC

Systemic wortmannin administration following intrapul-monary xenotransplantation of human NSCLC signifi-cantly increased mean survival in mice. After xenotrans-plantation of squamous cell carcinoma (KNS-62) sys-temic administration of 1 mg wortmannin per kilogramof bodyweight extended mean survival by 38% from23.6±7.3 days in untreated controls to 32.8±6.4 days(P<0.05) in the treatment group. In adenocarcinoma(Colo-699) bearing animals systemic wortmannin ad-ministration extended mean survival by 47% from18±2 days in untreated references to 26.5±2.1 days(P<0.01) in the treatment group (Fig. 3).

In addition to the extension of animals’ survival, toxicside effects due to wortmannin administration were ob-served. In five animals (36%) weight loss greater 10% ofbodyweight was observed, four (28%) had bloody diar-rhea or hemoglobinuria, in seven (50%) the fur becamedull, and in four (28%) physical activity was markedlyreduced. When toxic side effects were observed, medica-tion was interrupted until complete remission of symp-toms for up to 4 days. There were no deaths related towortmannin administration observed.

Discussion

We recently demonstrated that PI3K is overexpressed inhuman lung cancer [1]. PI3K has been shown to have apotential role in transduction of mitogenic signals in pro-liferating tumor cells [25]. This makes PI3K a potentialtarget for anticancer drug development because of itsrole as a component of growth factor and oncogene acti-vated signaling pathways [14]. Wortmannin has beendemonstrated effectively to block bradykinin-inducedPI3K kinase activity and also to abolish bradykinin-in-duced activation of the nuclear transcription factor κB

Fig. 2A, B Selective blocking of PI3K activity prior to subcutane-ous xenotransplantation caused significant delay in subcutaneoustumor growth. A Mean tumor volume in squamous cell carcinoma(KNS-62) 28 days after subcutaneous tumor induction measured27% compared to untreated references. B In adenocarcinoma(Colo-699) mean tumor volume was 22% of control tumors;*P<0.05, error bars SD

Fig. 3 Systemic administration of wortmannin significantly in-creased animals’ survival following intrapulmonary xenotransplan-tation of human NSCLC by 38% and 47%; *P<0.05, **P<0.01

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[26]. Blocking of PI3K activity by wortmannin is mostlikely due to covalent attachment at the electrophilic C-21 site [27]. PI3K-related kinases function in both cellcycle progression and DNA damage-induced cell cyclecheckpoints. Wortmannin is a potent inhibitor of DNA-dependent protein kinase and ataxia teleangiectasia mu-tated protein [28]. The PI3K downstream effector Akt/protein kinase B is a constitutively active kinase thatpromotes survival of NSCLC cells. Blocking of PI3K bywortmannin and thereby inhibiting activation of Akt/protein kinase B by phosphorylation decreases cellularsurvival in NSCLC [29]. In line with these observations,wortmannin has been observed significantly to inhibit invitro proliferation of NSCLC cells in correlation withconcentration. Concentrations employed were demon-strated previously to be selective for PI3K inhibition[30]; supraselective wortmannin concentrations (400 nM)caused an almost complete stop in proliferation. This isin accordance with Vemuri and Rittenhouse [31] andSchultz et al. [25], who observed in vitro significant in-hibition of proliferation of GC3 colon carcinoma cells,IGROV1 ovarian carcinoma cells, CHRF-288, andCCRF-CEM leukemia cells by wortmannin, although inother cell lines wortmannin failed to cause inhibition ofproliferation. This difference may be due to different lev-els of PI3K activity; as Moore et al. [32] first observed,PI3K is constitutively active in small-cell lung cancercells. In accordance with this we observed by immuno-histochemistry the overexpression of PI3K in KNS-62and Colo-699 NSCLC tumors. Inhibition of PI3K activi-ty by wortmannin markedly inhibits SCLC cell prolifera-tion and promotes cell cycle delay in G1 phase [32]. Thisis in accordance with our observation that in vitrowortmannin causes a cell cycle arrest but does not in-duce significant cytotoxicity or induction of apoptosis inhuman NSCLC cells. In line with this Miskimis et al.[33] observed that PI3K inhibitor wortmannin blocks mi-togenic activation of the transferrin receptor gene pro-moter in late G1, as the transferrin receptor is necessaryfor cells to progress through S phase.

The inhibition of NSCLC cell proliferation in vitroby the PI3K inhibitor wortmannin was reproduced in

vivo as a significant delay of subcutaneous tumorgrowth after to inhibition of PI3K activity bywortmannin prior to xenotransplantation. The markeddelay in tumor growth after subcutaneous xenotrans-plantation is in line with findings reported by Moore etal. [32], demonstrating that inhibition of PI3K activityby wortmannin inhibits anchorage-independent growthof SCLC cells and thereby their ability to survive andmetastasize in vivo. In addition to this, Schultz et al.[25] demonstrated inhibition of PI3K activity bywortmannin to inhibit growth of the murine C3H mam-mary carcinoma and the human BxPC3 pancreatic tu-mor in a xenograft model. These findings were repro-duced by Lemke et al. [17] in murine MCF-7 and C3Hmammary tumors. However, according to Schultz et al.[25], the ability of wortmannin to inhibit C3H tumorgrowth is not related to inhibition of tumor PI3K activi-ty, suggesting an additional site of action forwortmannin to inhibit tumor growth.

In the intrapulmonary xenotransplant model for hu-man NSCLC continuous wortmannin administration sig-nificantly extended animals survival by 38% and 47%.To ensure continued inhibition of PI3K activity a dose of1 mg wortmannin per kilogram of bodyweight was em-ployed. This dose was previously verified as efficient byDavol et al. [30].

In conclusion, the administration of wortmannin sig-nificantly inhibited proliferation of human NSCLC celllines in vitro. This effect was reproduced in vivo as a de-layed growth of subcutaneously xenotransplanted tumorsafter incubation of NSCLC cells with wortmannin. Sys-temic administration of wortmannin significantly extend-ed animals’ survival following intrapulmonary xeno-transplantation of human NSCLC cells. Inhibition ofPI3K activity by wortmannin appears a conceivable ex-planation of the observed effect. In line with the previ-ously demonstrated overexpression of PI3K in humanNSCLC [1], inhibition of PI3K activity appears as a po-tential target for treatment of human NSCLC. Systemictoxicity of wortmannin requires research for of improvedPI3K inhibitors with favorable pharmacological proper-ties.

References

1. Lin X, Boehle AS, Dohrmann P, Leuschner I, Schulz A, Kremer B, Faendrich F (2001) Overexpression ofphosphatidylinositol 3-kinase in humanlung cancer. Langenbecks Arch Surg386:293–301

2. Abraham RT (1996) Phosphatidylinosi-tol 3-kinase related kinases. Curr OpinImmunol 8:412–418

3. Carpenter CL, Cantley LC (1996)Phosphoinositide 3-kinase and the reg-ulation of cell growth. Biochim Bio-phys Acta 1288:M11–M16

4. Alimandi M, Wang LM, Bottaro D,Lee CC, Kuo A, Frankel M, Fedi P,Tang C, Lippman M, Pierce JH (1997)Epidermal growth factor and beta-cellulin mediate signal transductionthrough co-expressed ErbB2 and ErbB3receptors. EMBO J 16:5608–5617

5. Anderson SM, Burton EA, Koch BL(1997) Phosphorylation of Cbl follow-ing stimulation with interleukin-3 andits association with Grb2, Fyn, andphosphatidylinositol 3-kinase. J BiolChem 272:739–745

6. Carver RS, Mathew PM, Russell WE(1997) Hepatic expression of ErbB3 isrepressed by insulin in a pathway sen-sitive to PI-3 kinase inhibitors. Endo-crinology 138:5195–5201

7. Fukui Y (1991) Phosphatidylinositol-3kinase, a candidate for a second mes-senger producer. Tanpakushitsu Kakusan Koso 36:1901–1910

239

8. Gamett DC, Greene T, Wagreich AR,Kim HH, Koland JG, Cerione RA(1995) Heregulin-stimulated signalingin rat pheochromocytoma cells. Evi-dence for ErbB3 interactions withNeu/ErbB2 and p85. J Biol Chem270:19022–19027

9. Izuhara K, Feldman RA, Greer P, Harada N (1996) Interleukin-4 inducesassociation of the c-fes proto-oncogeneproduct with phosphatidylinositol-3 ki-nase. Blood 88:3910–3918

10. Izuhara K, Harada N (1996) Interleu-kin-4 activates two distinct pathwaysof phosphatidylinositol-3 kinase in thesame cells. Biochem Biophys ResCommun 229:624–629

11. Kim HH, Sierke SL, Koland JG (1994)Epidermal growth factor-dependent as-sociation of phosphatidylinositol 3-ki-nase with the erbB3 gene product.J Biol Chem 269:24747–24755

12. Matsuo T, Hazeki K, Tsujimoto N, Inoue S, Kurosu H, Kontani K, HazekiO, Ui M, Katada T (1996) Associationof phosphatidylinositol 3-kinase withthe proto-oncogene product Cbl uponCD38 ligation by a specific monoclo-nal antibody in THP-1 cells. FEBS Lett397:113–116

13. Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ, Wen SC,Zon G, Gewirtz AM, Perussia B, Calabretta B (1995) Phosphatidylinosi-tol-3 kinase activity is regulated byBCR/ABL and is required for thegrowth of Philadelphia chromosome-positive cells. Blood 86:726–736

14. Powis G, Berggren M, Gallegos A,Frew T, Hill S, Kozikowski A, Bonjouklian R, Zalkow L, Abraham R,Ashendel C (1995) Advances withphospholipid signalling as a target foranticancer drug development. ActaBiochim Pol 42:395–403

15. Arcaro A, Wymann MP (1993)Wortmannin is a potent phosphatidyl-inositol 3-kinase inhibitor: the role ofphosphatidylinositol 3:4,5-trisphos-phate in neutrophil responses. BiochemJ 296:297–301

16. Powis G, Bonjouklian R, BerggrenMM, Gallegos A, Abraham R, Ashendel C, Zalkow L, Matter WF,Dodge J, Grindey G (1994)Wortmannin, a potent and selective in-hibitor of phosphatidylinositol-3-ki-nase. Cancer Res 54:2419–2423

17. Lemke KE, Paine-Murrieta GD, TaylorCW, Powis G (1999) Wortmannin in-hibits the growth of mammary tumorsdespite the existence of a novelwortmannin-insensitive phosphatidyl-inositol 3-kinase. Cancer ChemotherPharmacol 44:491–497

18. Yano H, Nakanishi S, Kimura K, Hanai N, Saitoh Y, Fukui Y, NonomuraY, Matsuda Y (1993) Inhibition of his-tamine secretion by wortmanninthrough the blockade of phosphatidyl-inositol 3-kinase in RBL-2H3 cells.J Biol Chem 268:25846–25856

19. Takaki, T (1980) An epithelial cell line(KNS-62) derived from a brain metas-tasis of bronchial squamous cell carci-noma. J Cancer Res Clin Oncol96:27–33

20. Boehle AS, Dohrmann P, Leuschner I,Kalthoff H, Henne-Bruns D (2000) Animproved orthotopic xenotransplantprocedure of human lung cancer inSCID bg mice. Ann Thorac Surg69:1010–1015

21. Matzinger P (1991) The JAM test. Asimple assay for DNA fragmentationand cell death. J Immunol Methods145:185–192

22. Shibata S, Asano T, Ogura A, Hashimoto N, Hayakawa J, Uetsuka K(1997) SCID-bg mice as xenograft re-cipients. Lab Anim 2:163–168

23. Tomayko MM, Reynolds CP (1989)Determination of subcutaneous tumoursize in athymic (nude) mice. CancerChemother Pharmacol 24:148–154

24. Papaioannou VE, Fox JG (1993) Effi-cacy of tribromoethanol anesthesia inmice. Lab Anim Sci 43:189–192

25. Schultz RM, Merriman RL, Andis SL,Bonjouklian R, Grindey GB, Rutherford PG, Gallegos A, Massey K,Powis G (1995) In vitro and in vivo ac-tivity of phosphatidylinositol-3-kinaseinhibitor Wortmannin. Anticancer Res15:1135–1139

26. Pan ZK, Christiansen SC, Ptasnik A,Zuraw BL (1999) Requirement ofphosphatidylinositol 3-kinase activityfor bradykinin stimulation of NF-kap-paB activation in cultured human epi-thelial cells. J Biol Chem274:9918–9922

27. Norman BH, Shih C, Toth JE, Ray JE,Dodge JA, Johnson DW, Ruther-ford PG, Schultz RM, Worzall JF,Vlahos CJ (1996) Studies on the mech-anism of phosphatidylinositol 3-kinaseinhibition by Wortmannin and relatedanalogs. J Med Chem 39:1106–1111

28. Sarkaria JN, Tibbetts RS, Busby EC,Kennedy AP, Hill DE, Abraham RT(1998) Inhibition of phosphoinositide3-kinase related kinases by the radio-sensitizing agent Wortmannin. CancerRes 58:4375–4382

29. Brognard J, Clark AS, Ni Y, Dennis PA(2001) Akt/protein kinase B is consti-tutively active in non-small cell lungcancer cells and promotes cellular sur-vival and resistance to chemotherapyand radiation. Cancer Res61:3986–3997

30. Davol PA, Bizuneh A, Frackelton AR(1999) Wortmannin, a phosphoinosi-tide 3-kinase inhibitor, selectively en-hances cytotoxicity of receptor-direct-ed-toxin chimeras in vitro and in vivoAnticancer Res 19:1705–1713

31. Vemuri GS, Rittenhouse SE (1994)Wortmannin inhibits ser um-inducedactivation of phosphoinositide 3-kinaseand proliferation of CHRF-288 cells.Biochem Biophys Res Commun202:1619–1623

32. Moore SM, Rintoul RC, Walker TR,Chilvers ER, Haslett C, Sethi T (1998)The presence of a constitutively activephosphoinositide 3-kinase in small celllung cancer mediastes anchorage inde-pendent proliferation via a protein ki-nase B and p70s6k-dependent pathway.Cancer Res 58:5239–5247

33. Miskimins WK, King F, Miskimins R(1997) Phosphatidylinositol 3-kinaseinhibitor Wortmannin blocks mitogenicactivation of the transferrin receptorgene promoter in late G1. Cell GrowthDiffer 8:565–570