Transcriptional activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory ...processes, including cell cycle progression (p21/WAF1), DNA repair (GADD45) and apoptosis (bax). Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.
The p53 tumor suppressor protein is a transcription factor with sequence-specific DNA binding activity that is thought to be important for the growth-inhibitory function of p53. DNA binding appears ...to require activation of a cryptic form of p53 by allosteric mechanisms involving a negative regulatory domain at the carboxyl terminus of p53. The latent form of p53, reactive to the carboxyl-terminal antibody PAb421, is produced in a variety of eukaryotic cells, suggesting that activation of p53 is an important rate-limiting step in vivo. In this report we provide evidence that phosphorylation of serine 378 within the carboxyl-terminal negative regulatory domain of the human p53 protein by protein kinase C correlates with loss of PAb421 reactivity and a concomitant activation of sequence-specific DNA binding. These effects are reversed by subsequent dephosphorylation of the protein kinase C-reactive site by protein phosphatases 1 (PP1) and 2A (PP2A), which restore the reactivity of p53 to PAb421 and regenerate the latent form of p53 lacking significant DNA binding activity. Thus, p53 is subject to both positive and negative regulation by reversible enzymatic modifications affecting the latent or active state of the protein, suggesting a possible mechanism for the regulation of its tumor suppressor function.
Loss of genetic material on the short arm of chromosome 17 is observed in approximately 40% of human astrocytomas (WHO grades II and III) and in approximately 30% of cases of glioblastoma multiforme ...(WHO grade IV). Previous studies of glioblastoma multiforme have shown that the p53 gene, located on the short arm of chromosome 17, is frequently mutated in these glioblastomas. To explore whether lower-grade astrocytomas are also associated with corresponding mutations of the p53 gene, we have investigated a series of 22 human astrocytomas of WHO grades II and III both for loss of heterozygosity on chromosome 17p and for p53 mutations. Mutations in the conserved regions of the p53 gene were identified by single strand conformation polymorphism analysis of exons 5, 6, 7, and 8 and were verified by direct DNA sequencing of the polymerase chain reaction products. p53 mutations were observed in 3 of 8 grade II astrocytomas and 4 of 14 grade II astrocytomas. In all 22 tumors, allelic loss of the short arm of chromosome 17 was investigated by restriction fragment length polymorphism analysis. One-half of the grade II astrocytomas (4 of 8) and grade III astrocytomas (7 of 14) exhibited allelic loss on chromosome 17p. Mutations in the p53 gene were exclusively observed in tumors with allelic loss on 17p. Our results show that p53 mutations are not restricted to glioblastoma multiforme and may be important in the tumorigenesis of lower-grade astrocytomas and that p53 mutations in lower-grade astrocytomas are associated with loss of chromosome 17p. These findings are consistent with a recessive mechanism of action of p53 in WHO grade II and III astrocytoma tumorigenesis.
The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used ...to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, III and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.
Previous studies have shown frequent allelic losses of chromosomes 9p, 10, 17p, and 22q in glial tumors. Other researchers have briefly reported that glial tumors may also show allelic losses of ...chromosome 19, suggesting a putative tumor suppressor gene locus on this chromosome (D. T. Ransom et al., Proc. Am. Assoc. Cancer Res., 32:302, 1991). To evaluate whether loss of chromosome 19 alleles is common in glial tumors of different types and grades, we performed Southern blot restriction fragment length polymorphism analysis for multiple chromosome 19 loci in 122 gliomas from 116 patients. Twenty-nine tumors had loss of constitutional heterozygosity of 19q, and four tumors had partial deletions of 19q. Allelic losses on 19q were restricted to grade III anaplastic astrocytomas (4/9) and grade IV glioblastomas (11/46), grade II oligodendrogliomas (2/5) and grade III anaplastic oligodendrogliomas (2/2), and grade II (5/8) and grade III (5/7) mixed oligoastrocytomas. These data demonstrate genetic similarities between astrocytomas, oligodendrogliomas, and mixed glial tumors and indicate the presence of a glial tumor suppressor gene on chromosome 19q.
Neurofibromatosis type I (NF1) is a hereditary tumor and developmental disorder whose defective gene was cloned previously. The protein product of the NF1 gene, neurofibromin, contains a domain that ...shows significant sequence homology to the known catalytic domains of mammalian Ras GTPase-activating proteins (GAP) and the yeast IRA1 and IRA2 proteins. This homologous region of neurofibromin has been shown to exhibit GAP activity toward Ras proteins. Malignant schwannoma cell lines from NF1 patients contain normal levels of GAP and nonmutated Ras proteins but barely detectable levels of neurofibromin, based on genetic mutations in the NF1 gene. Because these cells contain constitutively activated Ras.GTP, it has been proposed that neurofibromin may be the sole negative regulator of Ras in these cells. Overall, these results have implied an important role of the Ras signaling pathway in NF1 malignant schwannomas. Recently, several laboratories have developed small molecule inhibitors of Ras function that inhibit the enzyme farnesyltransferase (FT). FT-mediated post-translational farnesylation of Ras proteins is absolutely necessary for Ras function since this modification is required for the anchoring of Ras proteins to the plasma cell membrane. Although previous studies have shown that FT inhibitors can block the growth of tumor cells carrying mutant Ras proteins, it remained unclear how this class of inhibitors would affect tumor cells such as in NF1, whose malignant growth appears to be mediated by up-regulation of wild-type Ras activity. Thus, in the current study, we investigated whether BMS-186511, a bisubstrate analogue inhibitor of FT, would inhibit the malignant growth properties of a cell line established from malignant schwannoma of an NF1 patient. Our results indicate that the malignant growth properties of ST88-14 cells, the most malignant cell line among several well-characterized NF1 cells, are inhibited by BMS-186511 in a concentration-dependent manner. Following treatment with BMS-186511, ST88-14 cells became flat, nonrefractile, were contact-inhibited, and lost their ability to grow in soft agar. In the drug-exposed cells, Ras proteins were prevented from FT-mediated membrane association. BMS-186511 was found to specifically inhibit FT, but not geranylgeranyltransferase I, a closely related enzyme. Thus, it is conceivable that FT inhibitors may ultimately become the first generation of drugs against the malignant phenotype in NF1 based on rational insights into the mechanism of action of neurofibromin.
A molecular genetic approach employing polymorphic DNA markers has been used to investigate the role of chromosomal aberrations in meningioma, one of the most common tumors of the human nervous ...system. Comparison of the alleles detected by DNA markers in tumor DNA versus DNA from normal tissue revealed chromosomal alterations present in primary surgical specimens. In agreement with cytogenetic studies of cultured meningiomas, the most frequent alteration detected was loss of heterozygosity on chromosome 22. Forty of 51 patients were constitutionally heterozygous for at least one chromosome 22 DNA marker. Seventeen of the 40 constitutionally heterozygotic patients (43%) displayed hemizygosity for the corresponding marker in their meningioma tumor tissues. Loss of heterozygosity was also detected at a significantly lower frequency for markers on several other autosomes. In view of the striking association between acoustic neuroma and meningioma in bilateral acoustic neurofibromatosis and the discovery that acoustic neuromas display specific loss of genes on chromosome 22, we propose that a common mechanism involving chromosome 22 is operative in the development of both tumor types. Fine-structure mapping to reveal partial deletions in meningiomas may provide the means to clone and characterize a gene (or genes) of importance for tumorigenesis in this and possibly other clinically associated tumors of the human nervous system.
Glioblastoma multiforme is a clinically and histologically heterogeneous lesion; however, to date, it has not been possible to subdivide glioblastomas on a clinical, histopathological or biological ...basis. Previous studies have demonstrated that loss of portions of chromosomes 10 and 17 and amplification of the epidermal growth factor receptor (EGFR) gene are the most frequent genetic alterations in glioblastoma. We therefore examined 74 glioblastomas from 67 patients for loss of heterozygosity on chromosomes 10 and 17, and for amplification of the epidermal growth factor receptor gene, to determine whether glioblastomas can be subtyped on a genetic basis. Using Southern blot analysis we were able to detect different patterns of genomic alterations. Eighteen of 67 informative patients were characterized by a loss of heterozygosity on the short arm of chromosome 17 in the tumor tissue. Forty‐five of 64 informative patients showed a loss of heterozygosity on chromosome 10. Amplification of the epidermal growth factor receptor gene was noted in 25 of 67 patients and was restricted to those glioblastomas that had lost portions of chromosome 10. Epidermal growth factor receptor gene amplification occurred significantly more often in patients without chromosome 17p loss than in patients with chromosome 17p loss (p = 0.01). In addition, those glioblastomas with a loss of chromosome 17p occurred in patients significantly younger than those with glioblastomas characterized by EGFR gene amplification (p = 0.001). These data emphasize the genetic heterogeneity of glioblastoma and suggest the division of glioblastoma into genetic subsets.
Overexpression of P-glycoprotein has been associated with a worse prognosis for some groups of patients not receiving chemotherapy. Recently, it has been demonstrated that in vitro both c-Ha-Ras ...overexpression and mutant p53 overexpression do activate the MDR1 gene (also known as PGY1) in murine NIH 3T3 cells. This direct connection between oncogenic activation, antioncogenic malfunctioning (presence of mutant instead of wild-type p53 protein), and MDR1 gene expression constitutes a fundamental conceptual model that could provide an explanation for the obscure prognostic role, in the absence of chemotherapy, of the MDR1 gene.
Our goal was to test whether the relationship between MDR1 (P-glycoprotein) expression, oncogenic activation, and mutant p53 protein expression demonstrated in vitro is also reproducible in vivo for two groups of human gynecologic tumors.
Fifty tumor specimens (31 mammary, 11 endometrial, and eight cervical) were analyzed. They had been obtained from previously untreated patients. Aliquots of these specimens had been frozen and stored at -70 degrees C since surgical collection or routinely fixed in formalin and embedded in paraffin. DNA was extracted from routinely fixed specimens for single-strand conformation polymorphism (SSCP) analysis. Immunohistochemical techniques were used on frozen material to determine: 1) P-glycoprotein expression using two different monoclonal antibodies (c219 and JSB1); 2) HER-2/neu (c-erb-B2; also known as ERBB2) expression using the NCL-CB11 monoclonal antibody; and 3) mutant p53 protein expression using the PAb 1801 monoclonal antibody. Polymerase chain reaction (PCR)-SSCP was used to confirm recognition of the mutated isoform of p53. Endometrial and cervical carcinomas were studied by both PCR-SSCP DNA analysis and immunohistochemical analysis. Only when there was full concordance between both methods were endometrial and cervical tumors considered to express mutant p53.
A statistically significant (P = .009; Fisher's exact test) association between HER-2/neu and MDR1 expression was found for the more aggressive form of inoperable, locally advanced mammary carcinoma. Expression of HER-2/neu or mutant p53 was similar in both tumor groups studied--mammary carcinoma with a low basal expression of P-glycoprotein compared with endometrial and cervical carcinomas with significantly (P = .0002; chi-square test) higher levels of expression.
The highly statistically significant coexpression of P-glycoprotein and HER-2/neu took place only in the subgroup of aggressive, locally advanced, inoperable mammary carcinomas, whereas no statistically significant association could be found for operable tumors. No association between mutant p53 expression and MDR1 activation was found in the human tumors analyzed.
The gene for the hereditary disorder neurofibromatosis type 2 (NF2), which predisposes for benign CNS tumors such as vestibular schwannomas and meningiomas, has been assigned to chromosome 22 and ...recently has been isolated. Mutations in the NF2 gene were found in both sporadic meningiomas and vestibular schwannomas. However, so far only 6 of the 16 exons of the gene have been analyzed. In order to extend the analysis of an involvement of the NF2 gene in the sporadic counterparts of these NF2-related tumors, we have used reverse transcriptase-PCR amplification followed by SSCP and DNA sequence analysis to screen for mutations in the coding region of the NF2 gene. Analysis of the NF2 gene transcript in 53 unrelated patients with meningiomas and vestibular schwannomas revealed mutations in 32% of the sporadic meningiomas (n = 44), in 50% of the sporadic vestibular schwannomas (n = 4), in 100% of the tumors found in NF2 patients (n = 2), and in one of three tumors from multiple-meningioma patients. Of the 18 tumors in which a mutation in the NF2 gene transcript was observed and the copy number of chromosome 22 could be established, 14 also showed loss of (parts of) chromosome 22. This suggests that in sporadic meningiomas and NF2-associated tumors the NF2 gene functions as a recessive tumor-suppressor gene. The mutations detected resulted mostly in frameshifts, predicting truncations starting within the N-terminal half of the putative protein.
PDF
