[CANCER RESEARCH 55. 2691-2695, June 15. 1995]

Expression of Peripheral-Type Benzodiazepine Receptor and Diazepam BindingInhibitor in Human Astrocytomas: Relationship to Cell Proliferation1

Helena Miettinen, Juha Kononen, Hannu Haapasalo, Paul! Helen, Paul! Sallinen, Tero Harjuntausta, Heikki Helin,and Hannu Alho2

Laboratory of Nettrohiology, Medical School, University of Tampere ¡H.M., T. H., H. A.j, and Departments of Pathology []. K., H. Ha., P. S.. H. He.I and Neurosurgery¡P.H.I, Tampere University Hospital, Medisiinarinkatu 3. 33101 Tampere. Finland

ABSTRACT

The expression of peripheral-type benzodiazepine receptor (PBR) and

diazepam binding inhibitor (DBI) were studied in human astrocytic tumors using immunocytochemistry and in situ hybridization. Both PBRand DBI were prominently expressed in neoplastic cells, whereas in normal brain their amount was low or undetectable. Immunocytochemicaldouble staining demonstrated that PBR and DBI were present in the samecells, suggesting that DBI may act in an autocrine manner in these cells.Analysis of 86 cases showed that PBR expression was statistically significantly associated with tumor malignancy grade (P = 0.004) and the

proliferative index as determined by immunocytochemistry with theMUM antibody (P = 0.004). Patients having tumors with high levels ofPBR-immunoreactive cells had a shorter life expectancy than patientswhose tumors showed lower PBR contents (/' = 0.024). In conclusion,

these results show that PBR expression is higher in neoplastic cells than innormal brain tissue. They also suggest that PBR immunocytochemistrymight be useful in evaluating malignancy in brain tumors.

INTRODUCTION

Two classes of benzodiazepine receptors have been identified inmammalian tissues. The central-type benzodiazepine receptor is lo

cated on the neuronal plasma membrane and is closely linked toGABAA receptors (1). PBRs3 form a unique class of receptors that

have pharmacologically different properties and that are probably notrelated in functional terms to central-type benzodiazepine receptors

(reviewed in Refs. 2 and 3). Although they are found in almost alltissues, the density of PBRs varies. In the central nervous system, theoverall PBR content is low, except in the cerebellum and spinal cordwhere distinct expression is detected (4-6). In the brain it appears that

PBRs are predominantly localized to glial cells (4, 5). Because of theirsubcellular localization to mitochondria, PBRs are often referred to asmitochondrial benzodiazepine receptors (7). However, recent reportsshow that other subcellular localizations of PBRs are also possible (8,9). The functional significance of PBRs and their effector mechanismsare not yet clear, but recent studies have suggested that they areinvolved in a number of functions such as mitochondrial cholesteroltransport, mitochondrial and cell proliferation, immune response, andmodulation of voltage-dependent calcium channels (10, 11).

A polypeptide capable of displacing benzodiazepine binding fromboth central-type and peripheral-type receptors has been purified from

the brain and liver of different species. Originally, the polypeptidewas designated as DBI (reviewed in Ref. 12). DBI is probablyinvolved in the regulation of multiple biological processes such asstimulation of cell growth, stimulation of steroidogenesis, and inhibition of glucose-induced insulin secretion from the pancreas

Received 1/3/95; accepted 4/18/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported by grants from the Sigrid Juselius Foundation, Academy of

Finland. Finnish Cancer Society, and Medical Research Fund of Pirkanmaa.2 To whom requests for reprints should be addressed.' The abbreviations used are: PBR, peripheral-type benzodiazepine receptor; DBI.

diazepam binding inhibitor; PBR-L1, PBR-like ¡mmunoreactivity.

(reviewed in Refs. 10 and 12). The polypeptide has also been purifiedby its ability to bind long chain acyl-CoA esters and accordinglyentitled to acyl-CoA-binding protein (13). DBI expression seems to be

restricted to special cell types in a given organ. In the brain, thehighest levels of DBI immunoreactivity have been detected in ependy-

mal and glial cells, although neurons also contain DBI. High concentrations of DBI are present in circumventricular organs and cerebellum,but not in the normal cerebral cortex (14).

In clinical settings it would be extremely useful to have biologicalmarkers that were capable of categorizing histologically similar astrocytic neoplasms into different prognostic subsets. PBRs are foundin low concentrations in normal brain, but there is increasing evidencethat PBRs are abundant in brain tumors. Several studies (15-20) have

pointed at increased binding of benzodiazepine ligands in differentbrain tumors. Although the biological functions of PBRs in braintumors remain unclear, it has been suggested that assaying benzodiazepine binding could be used in detecting tumors from normal braintissue or even directing therapies at tumor cells (16-20). Although we

have data on benzodiazepine binding to brain tumors and researchdemonstrating PBR expression in tumor cell lines of glial origin, thereare no earlier reports on PBR expression in astrocytic tumors. In thisstudy, we have used immunocytochemistry and in situ hybridizationto study the expression of PBRs and its putative endogenous ligandDBI in 86 astrocytoma cases and compared PBR expression to patientsurvival, histological grade, and the expression of the cell proliferationmarker MIB-1 used in previous studies (21, 22).

MATERIALS AND METHODS

The patients had been operated on at Tampere University Hospital,(Tampere, Finland) between February 1988 and February 1992. Of the previously described total material of 105 cases (23), 19 astrocytic tumors wereexcluded from this study because these tumor specimens were small and/ornecrotic, or they were not collected in the primary brain tumor operation. Thisleft us with 86 patients (median age, 47 years; mean ±SD, 44 ±18; range,5—77years; 21 females and 65 males). All of the patients underwent surgery

following the uniform practice of radical resection of the primary tumorwhenever possible. Most of the patients with high-grade (grades III-IV)

astrocytomas also received postoperative radiation therapy as did the patientswith low-grade (grades I—II)but recurrent tumors. All patients were followed

up using computed tomography scanning or magnetic resonance imaging every6—12months. Some of the patients with malignant gliomas received chemotherapy [l-(2-chloroethyl)-3-cyclohexyl-l-nitrosoureaj.

The tumors had been classified and graded by two pathologists according tothe WHO nomenclature (24). The material included 9 pilocytic astrocytomatumors of grade I, 19 tumors of grade 11,21 anaplastic astrocytomas of gradeIII, and 37 glioblastomas of grade IV. Thus, 68% of the tumors were highlymalignant. The follow-up period was at least 24 months.

Immunocytochemistry. The specimens were fixed in 4% phosphate-buff

ered formaldehyde (median fixation time less than 24 h). They were thenprocessed into paraffin blocks, and representative 5-/j.m sections were cut andtaken on poly-l.-lysine-coated slides. The slides were dried at 37°Covernight

and stored at room temperature until used. The sections were dewaxed, treatedwith solution containing methanol and 1.5% hydrogen peroxide, and hydrated.To reduce nonspecific immunostaining, the sections were incubated in PBS(pH 7.4) containing 10% BSA for 30 min. For visualization of PBR, the

2691

Research. on October 1, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

PBR EXPRESSION IN HUMAN ASTROCYTOMAS

samples were incubated for 16 to 22 h either with a polyclonal (1:500) or amonoclonal (1:50) PBR antibody (kindly donated by Dr. Krueger; Ref. 11) at4°C.For demonstration of DBI, a polyclonal rabbit DBI antibody was used

(1:4000; Ref. 14). The slides were then incubated with biotinylated goatanti-rabbit antibody (1:400; Vector Laboratories, Burlingame, CA) and avidin-

biotin complex for 30 min each. The immunoreaction was visualized byincubating the sections with 0.025% diaminobenzidine-0.01% hydrogen per

oxide for 5 min, or for fluorescence double labeling either with rabbit or mouseF1TC- or rhodamin-conjugated antisera. All of the antibodies were diluted inPBS containing 1% BSA and 0.3% Triton-X 100. After staining the sections

were embedded in Aquamount mountain!. The specificity of the PBR antibodyhas been previously studied using immunoprecipitation and Western blotting(11). MIB-1 immunocytochemistry was performed as described elsewhere

(21). For antigen retrieval, citrate buffer (pH 6.0) was used in a microwave

processing. The sections were treated four times for 5 min at 850 W maximumand 300 W power in a household microwave oven, after which the sectionswere allowed to cool in a buffer for 30 min. For the immunostaining of theKi-67 antigen, the mAb MIB-1 (IgGl; Immunotech SA, Marseille, France)

was used at a 1:40 concentration. The sections were incubated overnight at+4°C, and the primary antibody was visualized using the streptavidin-biotin

technique (Zymed Lab, Inc., CA). The counterstaining was done using 0.4%ethyl green in acetate buffer for 15 min.

Quantitation of Immunocytochemistry. Unware of the histológica!grades of the samples, two observers (J. K. and H. A.) evaluated the stainingsunder a light microscope and divided the cases into four different groups (0-3)

on the basis of the amount of PBR immunopositivity. Several microscopicvisual fields were analyzed (X 200). Only neoplastic cells were included in theanalysis. Necrotic and hemorrhagic areas and the section borders were omitted.

ï~I '

•¿�•\t¡'••!' /

'"I

1A Gr

" »

'* «*

BT'". ^

Griv

Fig. 1. Immunocytochemical demonstration ofPBR-L1 in high-grade malignant astrocytoma(grade IV) and in low-grade astrocytoma (grade I).

A, photomicrograph of grade I astrocytoma stainedwith PBR antiserum. Virtually no immunoreactivecells can be observed. B, grade IV astrocytomashowing high amount of PBR-LI cells. C and £>,higher magnifications of grade I astrocytoma (C)and grade IV astrocytoma (D) stained with thePBR antibody. D, higher magnification of the areain the outlined box in B. Arrows, cells expressingPBR immunoreactivity, which is granular and present mainly in the cytoplasm. E, H & E-stained

section of grade I astrocytoma. F, photomicrographof H & E-stained slide of a highly malignant astro-

cytic tumor (grade IV) demonstrating polymorphicnuclei and mitotic figures. Bars, 75 {¿m(A and B);20 jam (C-F).

*.*

C•¿�:

<;^2692

Research. on October 1, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

PER EXPRESSION IN HUMAN ASTROCYTOMAS

MIB-1 immunocytochemistry was quantitated using a computer-based imageanalysis system (CAS-200 Software; Becton Dickinson, Mountain View, CA).The microscope-based system is equipped with two cameras that convert the

image of immunopositive areas (brown) and immunonegative areas (green) innuclei for computer processing. The proliferation index is the percentageexpressing the ratio between the brown and the green images. For the analysis,20 microscope fields per each tumor were examined using x 400.

In Situ Hybridization. Fresh unfixed tissue obtained during the operationwas frozen and stored in liquid nitrogen until used. Sections were cut at —¿�20°C

and thawed onto Superfrost Plus (Menzel, Germany) slides. An oligonucleo-tide probe directed against PER mRNA (nucleotides 323-368, GenBankaccession number M36035; Ref. 23) was labeled at the 3' end with 35S-dATP

(DuPont-N England Nuclear Research Products, Boston, MA) using terminal

deoxynucleotidyltransferase (Amersham Int., Buckinghamshire, United Kingdom). A detailed description of the hybridization method has been publishedelsewhere (25). Briefly, the sections were hybridized at 42°Cfor 18 h with1 x 7 IO7 cpm/ml of the probe, washed four times for 15 min each in 1 X SSCat 55°C,and while in the final rinse they were left to cool to room temperature

(approximately 1 h). After exposing the autoradiograph films (Amershamß-max;Amersham Int.) for 3 weeks, they were developed using LX24 devel

oper and AL4 fixative (Kodak, Rochester, NY). Alternatively, the slides weredipped in Kodak NTB2 nuclear track emulsion and exposured for 3 weeks. Thedipped sections were counterstained with hematoxylin.

Statistical Methods. The statistical significance of the association of PBRexpression with tumor malignancy grade was determined with the x* test. To

test the relationship of PBR with MIB-1 expression or with tumor cellularityand patient age, the Mann-Whitney U and ! tests were used, respectively. The

statistical significance of survival differences between patient groups wasdetermined using the log rank test. All statistical analyses were performedusing SPSS for Windows software (SPSS Inc., Chigaco, IL).

Multivariate survival analysis was performed using the stepwise Coxregression model to evaluate the predictive power of the variables.

RESULTS

In low-grade astrocytomas only very few cells expressing PBR-likeimmunoreactivity (LI) were observed (Fig. 1A). By contrast, high-grade astrocytic neoplasms contained large numbers of PBR-LI ex

pressing cells (Fig. IB). The PBR immunostaining was granular, andunder higher magnification it was found to localize both around thenucleus and in the cytoplasm of the cells (Fig. ID). The immunocy-

tochemical results were confirmed by in situ hybridization with thePBR oliconucleotide probe; in normal cortex and low-grade astrocy

tomas no mRNA was present (Fig. 2/4), whereas in glioblastomas astrong hybridization signal was observed (Fig. 2B). Astrocytic tumorsof grades II and III showed a moderate amount of PBR mRNA (datanot shown). PBR-LI levels were significantly (P = 0.0036) associated

with the histológica! grade of the tumor. Grade I tumors showed lowor undetectable levels of PBR immunoreactivity, whereas grade II andIII tumors had considerable amounts of PBR-LI cells. There was no

significant difference in PBR expression between grade II and gradeIII tumors, and some samples were completely devoid of PBR immunoreactivity. PBR-LI was quite uniformly expressed in grade IVglioblastomas, although five cases showed very little PBR-LI (summarized in Table 1). The amount of PBR-LI was increased indepen

dently of cell density (t test). A clear association was observed withthe proliferative index as determined by immunocytochemistry withthe MIB-1 antibody (P = 0.0039, Mann-Whitney U test), demonstrating that PBR expression is related to cell proliferation. PBR-LI

expression was significantly associated with patient age but not sex(t test). Survival analysis indicated that PBR expression was associated with poor survival, and patients whose tumors did not expressPBR-LI lived longer (P = 0.024, Fig. 3). Survival analysis withingrade II tumors (n = 19) showed that patients with high PBR

expression had a tendency for shorter survival time, although thisfinding was not statistically significant (data not shown). Analysis

-'

r.

•¿�•«

Fig. 2. Photomicrograph of in situ hybridizations with the PBR oligonucleotide probe.A, virtually no silver grains observed in normal brain cortex; B, intensive hybridizationsignal present over malignant cells in grade IV astrocytoma; C, higher magnification ofemulsion autoradiogram with PBR probe hybridized to glioblastoma section. Clusters ofsilver grains can be observed around malignant cells. Burs, 10 firn (A and B); l /im (C).

within grade IV tumors (n = 37) revealed no differences in survival

time. In multivariate survival analysis, the predictive power ofPBR-LI was lower than that of the histological grade, patient age, andMIB-1 immunocytochemistry, which all were independent prognos-

ticators (data not shown).DBI-LI was observed in the cytoplasm of some cells in normal

brain. The expression of DBI-LI was higher in malignant astrocytomas than in normal brain or low-grade astrocytomas (data not shown).

The majority of the cells expressing DBI were also found to contain

2693

Research. on October 1, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

PBR EXPRESSION IN HUMAN ASTROCYTOMAS

Table 1 Association of PBR-LI with histological grades (¡-IV)of astrocylomas

PBR-LI0Grade

IGradeIIGradeIIIGradeIVNo.

of cases044301113%122151011%232481720%301113244856%No.

ofcases9(10%)19

(23%)21(24%)37

(43%)86(100%)100%r"P<0.0012

" The tumors were divided into the following classes on the basis of PBR content: class

0, tumors with no PBR-LI expression; class 1, tumors showed very little or no PBR-LI;class 2, tumors with high or moderate number of PBR-LI cells; and class 3, astrocytomasexhibited very high amount of PBR-LI.

' X2 test was used to assess the probability of randomly attaining these results. For theX2 test, the tumors were regrouped into PBR-LI-negative (classes 0 and 1) and PBR-LI-

positive (classes 2 and 3) groups.

PBR-LI

0 6 12 18

Survival Time (months)

Fig. 3. Cumulative survival of patients with low and high levels of PBR-LI expression.For this analysis patients were regrouped into low PBR-LI (classes 0 and 1) and highPBR-LI (classes 2 and 3) groups. Patients with high amounts of PBR-LI cells show atendency to poor survival when compared to patients with low PBR-LI levels(P = 0.0239).

PBR in an immunocytochemical double-staining procedure. However, there were also many PBR-immunoreactive cells lacking DBI

immunoreactivity (Fig. 4).

DISCUSSION

In this study we have examined the expression of PBR in 86astrocytomas by immunocytochemistry and by in situ hybridization.We observed a clear association between the amount of PBR and themalignancy of astrocytomas, whereas normal brain expressed unde-

tectable levels of PBR mRNA and protein. These results are consistent

with previous findings which have suggested the presence of benzo-diazepine receptors in brain tumors and demonstrated increased ben-

zodiazepine binding in astrocytomas and other gliomas (15), as wellas expression of PBRs in glioma cell lines (19). According to ourresults there is a statistically significant association between PBR-LI

and the histological grade of the tumor. None of the grade I pilocyticastrocytomas expressed intensive PBR-LI, and most of the pilocyticastrocytomas showed no PBR-LI. There was far greater variety in the

PBR content of grade II and III tumors. This may be an indication ofoverall heterogenity in these anaplastic astrocytomas. PBR-LI was

more uniformly expressed in grade IV glioblastomas. Taken togetherwith the association of high levels of PBR-LI with a high cellular

proliferation rate, our results suggest that high PBR expression is acharacteristic feature of malignant cells. To the best of our knowledge,there has been no research to explore the possible links between PBRcontent and patient survival. We observed that high PBR-LI is sig

nificantly associated with poor patient survival, suggesting that PBRexpression may be indicative of aggressive tumor growth. However,since PBR expression is also associated with pathological grade, it ispossible that correlation between PBR staining with survival couldsimply reflect the differences in survival between low- and high-gradetumors. Therefore, we performed survival analysis also within low-and high-grade subgroups. Within grade II tumors, those with high

PBR content showed a tendency toward poor survival, supporting theconcept that PBR expression may imply aggressive tumor growthindependently of pathological grade. Probably due to the low numberof patients (n = 19) this finding did not reach statistical significance.

It has been suggested that elevated benzodiazepine binding ¡nmalignant tissues might reflect increased cell density, and the rise inPBR expression in tumors has been questioned (15). The method ofimmunocytochemistry allows us to assess the histological features oftissue sections, and our study clearly shows that the amount of PBRis increased independently of cell density. This is confirmed by in situhybridization data, which suggest that the increased PBR content isdue to changes in PBR gene expression. Further studies are needed toestablish whether the mechanisms involved are increased transcrip-

tional activity or gene amplification. The latter seems unlikely in thatthe PBR gene is located in the ql3.3 region of chromosome 22 (26),and a recent study of nine malignant astrocytomas using comparativegenomic hybridization demonstrated no apparent amplifications inastrocytomas in this chromosomal region. On the contrary, deletionsin the ql3.3 region of chromosome 22 were found in five of nine cases(27), suggesting that this may be a common feature in astrocytomas.This is one possible explanation for the negative PBR-LI findings insome high-grade astrocytomas in our patient material.

Fig. 4. A, photomicrograph of an astrocytomasection stained with the PBR antibody. The immu-nostaining is visualized by bright fluorescent labeling, appearing white in this micrograph. B, immu-nofluorescence photomicrograph of an astrocytomasection stained with the DBI antibody. Arrows,cells expressing both PBR and DBI; arrowheads,cells containing PBR but devoid of DBI; openarrows, DBI-immunoreactive cells that do not

express PBR. Bar, 10 /im.

26"4

Research. on October 1, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

PBR EXPRESSION IN HUMAN ASTROCYTOMAS

An interesting discovery that we made was that DBI, which hasbeen suggested to function as an endogenous ligand for PBR, isexpressed in the same cells with PBR in astrocytomas. As in the caseof PBR, the amount of DBI was higher in malignant astrocytomasthan in normal brain. This is consistent with recent observationsindicating that DBI-LI and DBI mRNA levels are increased in ma

lignant astrocytomas (28). Colocalized expression of DBI and PBRsuggests that DBI may have autocrine functions. On the basis of theirexistence in glioma cell lines, Ferrarese et al. (15) proposed anautocrine model for DBI function. Taken together, our findings suggest the conclusion that the expression of both the receptor and theputative ligand are increased in brain tumors, indicating that thefunction of DBI acting through PBR may be accelerated in malignantcells. However, since it has not been possible to establish whichbiological functions DBI accomplishes through PBR, it may be thatthey perform different, possibly related tasks in these cells. Ourfindings of increased DBI expression in malignant cells which aremetabolically very active are in agreement with the possibility thatDBI plays a role in basal cell metabolism.

As yet we do not have a clear picture of the biological functions ofPBR. It has been shown that benzodiazepines affect the proliferationrate of C6 glioma cells in a dose-dependent manner (29), suggesting

a role for PBR in the mitotic processes. This is consistent with ourresults according to which PBR expression correlated statisticallysignificantly with the frequency of proliferating cells labeled with theantibody MIB-1. In vitro, benzodiazepines induce morphological

changes and increase the proliferation rate of mitochondria (29).Energy requirements are higher in proliferating cells, such as malignant cells in glioblastoma. One possible function for PBR could thusbe the regulation of mitochondria! replication. On the other hand, invitro studies have shown that PBRs are involved in neurosteroidbiosynthesis (reviewed in Refs. 10, 30, and 31). High expression ofPBRs in astrocytomas might thus be an indicator of increasedmetabolic activity and steroid biosynthesis in malignant cells.

This study shows that PBR expression is closely associated withcell proliferation and tumor malignancy. Furthermore, the patientswith tumors expressing high PBR levels showed a tendency for poorsurvival. Colocalization of DBI and PBR and elevated DBI expressionin malignant tumors suggest that DBI may act in an autocrine manner,possibly through the PBR present in the same cell.

ACKNOWLEDGMENTS

We thank Markku Pelto-Huikko for his help with in situ hybridization and

Meelis Kolmer for his helpful comments on the manuscript.

REFERENCES

1. Costa, E., Guidoni, A., Mao, C. C., and Suria, A. New concepts of the mechanism ofaction of benzodiazepines. Life Sci., 17: 167-186, 1975.

2. Krueger, K. E. Peripheral-type benzodiazepine receptors: a second site of action forbenzodiazepines. Neuropsychopharmacology, 4: 237-242, 1990.

3. Snyder, S. H., McEnery, M. W., and Verma, A. Molecular mechanisms of peripheralbenzodiazepine receptors., Neurochem. Res., /5: 119-123, 1990.

4. Anholt, R. R. H., Murphy, K. M. M., Mack, G. E., and Snyder, S. H. Peripheral-typebenzodiazepine receptors: autoradiographic localization in whole-body sections ofneonatal rats. J. Pharmacol. Exp. Ther., 233: 517-526, 1985.

5. Villiger, J. W. Characterization of peripheral-type benzodiazepine recognition sites inrat spinal cord. Neuropharmacology, 24: 95-98, 1985.

6. Genieri, D. R., Yamamura, H. I., and Wamsley, J. K. Autoradiographic localization

of "peripheral" benzodiazepine binding sites in rat brain and kidney. Eur. J. Pharma

col., 95: 329-330, 1985.7. Anholt, R. R. H., Pedersen, P. L., DeSouza, E. B., and Snyder, S. H. The peripheral-

type benzodiazepine receptor: localization to the mitochondrial outer membrane.J. Biol. Chem., 261: 576-583, 1986.

8. O'Beirne, G. W., Woods, M. J., and Williams, D. C. Two subcellular locations of

peripheral benzodiazepine-type acceptors in rat liver. Eur. J. Biochem., 188: 131-138,

1990.9. Oke, B. O., Suarez-Quian, C., Riond, J., Ferrerò, P., and Papadopoulos, V. Cell

surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenalcortex. Mol. Cell. Endocrinol., 87: R1-R6, 1992.

10. Papadopoulos, V. Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: biological role in steroidogenic cell function. Endocrinology, 14: 222-238, 1993.

11. Alho, H., Varga, V., and Krueger, K. E. Expression of mitochondrial benzodiazepinereceptor and its putative endogenous ligand diazepam binding inhibitor in culturedprimary astrocytes and C-6 cells: relation to cell growth. Cell Growth & Differ., 5:1005-1014, 1994.

12. Costa, E., and Guidoni, A. Diazepam binding inhibitor (DBI): a peptide with multiplebiological actions. Life Sci., 49: 325-344, 1991.

13. Knudsen, J., Mandrup, S., Rasmussen, J. T., Andreasen, P. H., Poulsen, F., andKristiansen, K. The function of acyl-CoA-binding protein (ACBP)/diazepam bindinginhibitor (DBI). Mol. Cell. Biochem., 123: 129-138, 1993.

14. Alho, H., Costa, E., Ferrerò, P., Fujimoto, M., Cosenza-Murphy, D., and Guidoni, A.Diazepam-binding inhibitor: a neuropeptide located in selected neuronal populationsof rat brain. Science (Washington DC), 229: 179-182, 1985.

15. Ferrarese, C., Appollonio, I., Frigo, M., Caini, S. M., Piolti, R., and Frattola, L.Benzodiazepine receptors and diazepam-binding inhibition in human cerebral tumors.Ann. Neurol., 26: 564-568, 1989.

16. Black, K. L., Ikezaki, K., Santori, E., Becker, D. P., and Vinters, H. V. Specifichigh-affinity binding of peripheral benzodiazepine receptor ligands to brain tumors inrat and man. Cancer (Phila.), 65: 93-97, 1990.

17. Ikezaki, K., Black, K. L., Toga, A. W., Santori, E. M., Becker, D. P., and Smith, M. L.Imaging peripheral benzodiazepine receptors in brain tumors in rats. In vitro bindingcharacteristics. J. Cerebr. Blood Flow Metab., 10: 580-587, 1990.

18. Ikezaki, K., Black, K. L., Santori, E. M., Smith, M. L., Becker, D. P., Payne, B. A.,and Toga, A. W. Three-dimensional comparison of peripheral benzodiazepine binding and histological findings in rat brain tumor. Neurosurgery, 27: 78-82, 1990.

19. Cornu, Ph., Benavides, J., Scatton, B., Hauw, J-J., and Philippon, J. Increase in 3(peripheral-type benzodiazepine) binding site densities in differential types of humanbrain tumours. Acta Neurochir., ¡19:146-152, 1992.

20. Ikezaki, K., and Black, K. L. Stimulation of cell growth and DNA synthesis byperipheral benzodiazepine. Cancer Lett., 49: 115-120, 1990.

21. Sallinen, P., Haapasalo, H. K., Visakorpi, T., Helen, P. T., Rantala, I. S., Isola, J. J.,and Helin, H. J. Prognostication of astrocytoma patient survival by Ki-67 (MIB1),PCNA and S-phase fraction using archival paraffin embedded samples. J. Pathol.,174: 275-282, 1994.

22. Montine, T. J., Vandersteenhoven, J. J., Aguzzi, A., Boyko, O. B., Dodge, R. K.,Kerns, B-J., and Burger, P. C. Prognostic significance of Ki-67 proliferation index insupratentorial fibrillary astrocytic neoplasms. Neurosurgery, 34: 674-679, 1994.

23. Haapasalo, H., Isola, J., Sallinen, P., Kalimo, H., Helin, H., and Rantala, I. Aberrantp53 expression in astrocytic neoplasms of the brain: association with proliferation.Am. J. Pathol., 142: 1347-1351, 1993.

24. Kleihues, P., Burger, P. C., and Scheithauer, B. W. Histological typing of tumours ofthe central nervous system. WHO. Berlin: Springer-Verlag, 1993.

25. Dagerlind, A., Friberg, K., Bean, A. J., and Hökfelt,T. Sensitive messenger RNAdetection using unfixed tissue-combined radioactive and non-radioactive in situhybridization histochemistry. Histochemistry, 98: 39-49, 1992.

26. Riond, J., Mattei, M. G., Kaghad, M., Dumont, N., Guillemot, J. C., Le Fur, G.,Caput, D., and Ferrara, P. Molecular cloning and chromosomal localization of ahuman peripheral-type benzodiazepine receptor. J. Biochem., 195: 305-311, 1991.

27. Schiock, E., Thiel, G., Lozanova, T., du Manoir, S., Meffert, M. C., Jauch, A.,Speicher, M. R., Nürnberg,P., Vogel, S., Janisch, W„et al. Comparative genomichybridization of human malignant gliomas reveals multiple amplification sites andnonrandom chromosomal gains and losses. Am. J. Pathol., 144: 1203-1218, 1994.

28. Alho, H., Kolmer, M., Harjuntausta, T., and Helen, P. Increased expression ofdiazepam binding inhibitor in human brain tumors. Cell Growth & Differ., 6:309-314, 1995.

29. Shiraishi, T., Black, K. L., Ikezaki, K., and Becker, D. P. Peripheral benzodiazepinesinduces morphological changes and proliferation of mitochondria in glioma cells. J.Neurosci. Res., 30: 463-474, 1991.

30. Guarneri, P., Papadopoulos, V., Pan, B., and Costa, E. Regulation of pregnenolonesynthesis in C6-2B glioma cells by 4'-chlorodiazepam. Proc. Nati. Acad. Sci. USA,

89: 5118-5122, 1992.

31. Papadopoulos, V.. Guarneri, P., Krueger, K. E., Guidoni, A., and Costa, E. Pregnenolone biosynthesis in C6—2Bglioma cell mitochondria: regulation by a mitochondrialdiazepam binding inhibitor receptor. Proc. Nati. Acad. Sci. USA, 89: 5113-5117,

1992.

2695

Research. on October 1, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from

1995;55:2691-2695. Cancer Res   Helena Miettinen, Juha Kononen, Hannu Haapasalo, et al.   Relationship to Cell ProliferationDiazepam Binding Inhibitor in Human Astrocytomas: Expression of Peripheral-Type Benzodiazepine Receptor and

  Updated version

  http://cancerres.aacrjournals.org/content/55/12/2691

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/55/12/2691To request permission to re-use all or part of this article, use this link

Research. on October 1, 2020. © 1995 American Association for Cancercancerres.aacrjournals.org Downloaded from