BCL-2 family protein expression in human malignant glioma: a clinical-pathological correlative study
Introduction
Median survival in malignant glioma is in the range of one year even with cytoreductive surgery, irradiation and adjuvant chemotherapy (Shapiro et al., 1989). Nitrosoureas such as BCNU, ACNU and CCNU are the most commonly used in malignant glioma patients and are included in most combination chemotherapy protocols currently evaluated. One such protocol is the PCV (procarbazine, CCNU, vincristin) protocol (Levin et al., 1989) that is clearly effective in anaplastic oligodendroglioma but has also shown effects in some glioma patients. The current NOA protocol of the German Cancer Society compares ACNU plus teniposide with ACNU plus cytarabine as adjuvant chemotherapy after surgery and irradiation. Although chemotherapy prolongs median survival only for a few weeks, the percentage of patients surviving for 18 months or more increases from 5% without chemotherapy to 20–25% with chemotherapy (Shapiro et al., 1989). Identifying this minority of patients that will significantly benefit from adjuvant chemotherapy by means of biological markers would be a great advance in the medical management of malignant glioma. There are probably multiple reasons why malignant gliomas are rather refractory to radiotherapy and chemotherapy, including aberrant growth signal transduction pathways, enhanced repair of DNA damage caused by therapeutic irradiation or chemotherapeutic agents, failure to achieve significant concentrations of cytotoxic agents in the tumour tissue, and prolonged administration of steroids (Weller et al., 1997).
Defects in the cascades required for physiological apoptotic cell death are now thought to play a central role in carcinogenesis and may contribute significantly to tumour cell resistance to therapeutic irradiation and cancer chemotherapy (Kerr et al., 1994, Reed, 1995, Reed et al., 1996). The regulation of susceptibility to apoptosis is under strict genetic control and involves several gene products, including the tumour suppressor proteins p53 and the retinoblastoma (RB) protein and members of the BCL-2 protein family. Changes in the expression of these gene products have also been reported in malignant gliomas (Weller, 1997), most extensively for p53 (Louis et al., 1993, Bögler et al., 1995) but also RB (Henson et al., 1994) and BCL-2 (Alderson et al., 1995, Weller et al., 1995).
The BCL-2 gene was first identified as a candidate oncogene in follicular B cell lymphomas which harbour the t(14;18)(q32;q21) translocation. The protein is physiologically expressed in mantle zone lymphocytes, thyroid gland epithelium and neurons. BCL-2 is the prototype member of a growing gene family that includes both antiapoptotic genes such as BCL-2, BCL-X and MCL-1 and proapoptotic genes such as BAX. BAX protein shares 21% amino acid identity with BCL-2, forms heterodimers with BCL-2 and other BCL-2-related proteins, and promotes cell death if present in sufficient quantity to form BAX homodimers. Deregulated expression of BCL-2 family genes is thought to play a major role both in the pathogenesis and the resistance to radiotherapy and chemotherapy in most human neoplasms (Reed, 1995, Reed et al., 1996, Weller, 1997). Thus, the levels of endogenous BCL-2 protein in malignant glioma cell lines predict resistance to cytotoxic CD95 antibodies (Weller et al., 1995), and forced expression of a murine BCL-2 transgene in glioma cells induces resistance to irradiation and BCNU (Weller et al., 1995). The balance between pro- and antiapoptotic proteins might be shifted toward higher resistance to apoptosis in tumour cells lacking wild-type p53 because p53 is a positive regulator of BAX and a negative regulator of BCL-2 (Miyashita et al., 1994).
p53 mediates most of its biological effects by transcriptional regulation of target genes such as waf/cip-1/p21, gadd-45, MDM-2, BAX or BCL-2 (Cox and Lane, 1995, Haffner and Oren, 1995). Activation of p53 induces a p21-dependent growth arrest which may allow for DNA repair in response to DNA damage (Waldman et al., 1995). Depending on the cellular context, e.g. when cells fail to arrest in G0/G1, p53 activity may precipitate apoptosis, an effect probably dependent on p53-induced activation of BAX gene expression (Miyashita et al., 1994). Loss of wild-type p53 status is the most common molecular abnormality in human neoplasms and may be the first event in the malignant progression of astrocytomas. The levels of normal p53 protein in untransformed cells are too low to be detected by immunohistochemistry (Hall and Lane, 1994). However, p53 protein accumulates and becomes detectable if the half life of the p53 protein is increased, e.g. and most commonly, by mutations of the p53 gene, or, alternatively, by stabilisation of wild type p53 protein, e.g. by enhanced expression of the MDM-2 protein, a negative feedback regulator of wild-type p53 activity (Reifenberger et al., 1993).
RB is the prototype tumour suppressor gene (Levine and Mommand, 1990, Levine, 1993, Kouzarides, 1995). RB-susceptible individuals carry a germ-line mutation in one RB allele and are highly susceptible to tumour formation elsewhere in the organism when the remaining wild-type RB allele acquires a mutation in a somatic cell. Such patients have a higher probability of developing tumours of connective tissue and brain. RB maintains a cell in G0/G1 by binding E2F which is required for S phase progression. RB is maintained in its active, hypophosphorylated form by p16-mediated inhibition of cyclin-dependent kinases, notably CDK-4. It has now been shown that the RB pathway is nonfunctional in the vast majority of malignant gliomas because of deletions of the p16 genes or enhanced expression of CDK-4 (Schmidt et al., 1994, Nishikawa et al., 1995, Ueki et al., 1996). In this regard, the clinical significance of wild-type p53 dependent proteolytic cleavage of RB in glioma cells remains to be investigated (Wagenknecht et al. (in press)).
In the present study, we have investigated the expression of several BCL-2 family proteins, p53 and RB in the tumour tissue of 20 glioma patients prior to irradiation and chemotherapy. Analysing the expression of BCL-2 family members in malignant gliomas in vivo was the major rationale. Further, we asked whether the parameters examined predicted response to therapy, that is, time to progression and overall survival.
Section snippets
Glioma specimens
All malignant gliomas included in this study were resected at the Department of Neurosurgery in Tübingen. The tumours were histologically analyzed by routine methods. Only WHO grade IV gliomas were included. The patients' age ranged from 17 to 69 years (n=20, median=50.5, SD(16)). All were primary resections. None of the patients had received prior radiotherapy or chemotherapy. The tumour specimens were a series of consecutive participants of the German NOA multicentre trial of adjuvant
Expression of proteins involved in the regulation of apoptosis in human malignant gliomas
Table 1 summarizes the results of the immunohistochemical analysis of BCL-2, BCL-X, MCL-1, BAX, p53 and RB in 20 human gliomas included in this study. BCL-2 was detected in 14 of 20 gliomas. Most typical was a granular pattern associated with diffuse cytoplasmic staining that was most prominent around the nuclear membrane (Fig. 1a). Normal astrocytes did not express BCL-2 protein. However, reactive astrocytes at the tumour margin stained intensely for BCL-2. Few neurons were BCL-2-positive in
Discussion
The reasons for the poor response of malignant glial neoplasms to current approaches of therapy are manyfold, as outlined above. The present study asked whether immunocytochemical examination of proteins involved in the regulation of susceptibility to apoptosis helps to identify the subset of malignant glioma patients that is likely to respond to chemotherapy. BCL-2 expression in malignant gliomas has been analyzed previously (Nakasu et al., 1994, Ellison et al., 1995a, Ellison et al., 1995b,
Acknowledgements
Supported by the Deutsche Forschungsgemeinschaft (We 1502/3-1) and the Fortüne-Programm of the University of Tübingen. The authors thank K. Dietz, PhD (Institute of Medical Biometry) for advice on statistical analysis.
References (40)
- et al.
The BAX gene maps to the glioma candidate region at 19q13, but is not altered in human gliomas
Cancer Genet. Cytogenet.
(1996) - et al.
Biochemical properties and biological effects of p53
Curr. Opin. Genet. Dev.
(1995) Functions of pRb and p53: what́s the connection
Trends Cell. Biol.
(1995)- et al.
Tumor suppressor genes: the p53 and retinoblastoma sensitivity genes and gene products
Biochim. Biophys. Acta.
(1990) - et al.
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 124-3-3 not BCL-XL
Cell
(1996) - et al.
MCL-1, a BCL-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions
Blood
(1997) - et al.
Human gliomas with wild-type p53 express BCL-2
Cancer Res.
(1995) - et al.
The p53 gene and its role in brain tumors
Glia
(1995) - et al.
BCL-X, a BCL-2 related gene that functions as a dominant regulator of apoptotic cell death
Cell
(1993) - et al.
Tumor suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage
Bioessays
(1995)
Prognostic indicators in a range of astrocytic tumors: an immunohistochemical study with Ki-67 and p53 antibodies
J. Neurol. Neurosurg. Psychiatry
Apoptosis in cerebral astrocytic tumors and its relationship to expression of the BCL-2 and p53 proteins
Neuropathol. Appl. Neurobiol.
Inactivation of BCL-2 by phosphorylation
Proc. Natl. Acad. Sci. USA
p53 in tumor pathology: can we trust immunohistochemistry?-revisited!
J. Pathol.
Lack of p16ink4 or retinoblastoma protein (pRB), or amplification-associated overexpression of cdk4 is observed in distinct subsets of malignant glial tumors and cell lines
Cancer Res.
The retinoblastoma gene is involved in malignant progression of astrocytomas
Ann. Neurol.
Apoptosis, its significance in cancer and cancer therapy
Cancer
Expression of BCL-2 in reactive and neoplastic astrocytes: lack of correlation with presence or degree of malignancy
J. Neurosurg.
Superiority of postradiotherapy adjuvant chemotherapy with CCNU, procarbazine and vincristine (PCV) over BCNU for anaplastic gliomas: NCOG 6G61 final report
Int. J. Radiat. Oncol. Biol. Phys.
The tumor suppressor genes
Annu. Rev. Biochem.
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