The p53 tumor suppressor targets a novel regulator of G protein signaling.

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. Herein we report on the identification of a novel p53 target gene (A28-RGS14) that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways.

A kindred with a variant of multiple endocrine neoplasia type 1 demonstrating frequent expression of pituitary tumors but not linked to the multiple endocrine neoplasia type 1 locus at chromosome region 11q13.

Acromegaly is uncommon in kindreds with multiple endocrine neoplasia type 1 (MEN1), whereas primary hyperparathyroidism (PHP) has the highest penetrance of any endocrinopathy. We report an unusual MEN1 kindred with frequent expression of pituitary tumors and a low penetrance of PHP. Four members were found to have disease: PHP in generation I, acromegaly (2 cases) in generation II, and hyperprolactinemia associated with a pituitary tumor in generation III. There was no evidence for PHP in 1 patient with acromegaly (age 60 yr), the patient with hyperprolactinemia and the pituitary tumor (age 22 yr), and 1 asymptomatic obligate carrier (age 50 yr). Screening of 26 members revealed the possible diagnosis of PHP in 1 family member in generation II and possible early acromegaly in 2 members of generation III with elevated serum concentrations of insulin-like growth factor I and insulin-like growth factor-binding protein-3 but normal patterns of pulsatile GH release. Although the predisposing genetic defect in typical MEN1 families has previously been mapped to chromosome location 11q13 without evidence of heterogeneity among the 87 families analyzed, linkage of disease in this family to the MEN1 region is unlikely based on haplotype analysis. Localization of the gene(s) responsible for disease in such atypical families may aid in the understanding of the pathogenesis of MEN1. In addition, further study of the earliest changes in patterns of pulsatile GH release in familial acromegaly may allow more insight into the pathogenesis and natural history of this disease.

Chromosome 17 allelic loss and NF1-GRD mutations do not play a significant role as molecular mechanisms leading to melanoma tumorigenesis.

Allelic loss in human cutaneous melanoma has been detected on chromosomes 1p, 6q, 9p, 10q, and 11q. Chromosome 17 contains important tumor suppressor genes such as p53, NM23, and neurofibromatosis type 1 (NF1), which have been implicated in melanoma tumorigenesis. The role of p53 has already been studied by a number of laboratories, showing contrasting results. In the present study, two restriction fragment length polymorphism (RFLP) probes for the NM23 and NF1 genes, together with five other RFLP and four variable number of tandem repeat chromosome 17 probes, were investigated at the loss of heterozygosity (LOH) level in a Southern blot-based assay. The NF1 gene was also tested for LOH by a polymerase chain reaction (PCR)-based approach in two different experiments, using a dinucleotide repeat polymorphic probe at locus D17S250 (17q11.2-q12), and an Alu probe intragenic to the NF1 gene (17q11.2). A PCR single-strand conformation polymorphism assay was included in the study for mutation detection at the NF1-GTPase-activating protein-related domain (GRD). A total of 68 melanocytic tumors were analyzed. LOH was detected in 9 of 87 informative cases (10%). LEW301 (17p11.2-pcen) presented the highest LOH frequency (22%). NM23 showed LOH in 17% of the informative cases, while NF1 did not show either LOH in the Southern blot- and PCR-based experiments or mutations at the NF1-GRD. These results are in concordance with those of previous smaller studies, but when compared with higher LOH frequencies obtained from other chromosomes, these findings indicate that the LOH values found in our study can most likely be attributed to background effect. Thus, chromosome 17 LOH is likely to play and unimportant role as a genetic event in melanoma tumorigenesis. Nevertheless, NF1 merits further study, since homozygous deletions have been detected at this locus in melanoma cell lines.

Development of highly potent inhibitors of Ras farnesyltransferase possessing cellular and in vivo activity.

Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a CA1A2X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were prepared where phenylalanine was replaced by (Z)-dehydrophenylalanine, 2-aminoindan-2-carboxylate, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic), and indoline-2-carboxylate. The greatest improvement in FT inhibitory potency was observed for the Tic derivative (IC50 = 1 nM); however, this compound was ineffective in blocking oncogenic Ras-induced transformation of NIH-3T3 fibroblast cells. A compound was prepared in which both the Cys-Val methyleneamine isostere and the Tic replacement were incorporated. This derivative inhibited FT with an IC50 of 0.6 nM and inhibited anchorage-independent growth of stably transformed NIH-3T3 fibroblast cells by 50% at 5 microM. Replacing the A1 side chain of this derivative with a tert-butyl group and replacing the X position with glutamine led to a derivative with an IC50 of 2.8 nM and an EC50 of 0.19 microM, a 26-fold improvement over (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-valyl]-1,2,3,4- tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine. This derivative, (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-tert-leucyl]-1,2,3,4 - tetrahydro-3-isoquinolinyl]-carbonyl]-L-glutamine, was evaluated in vivo along with (S*,R*)-N-[[2-[N-(2-amino-3- mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3- isoquinolinyl]carbonyl]-L-methionine methyl ester for antitumor activity in an athymic mouse model implanted ip with H-ras-transformed rat-1 tumor cells. When administered by injection twice a day at 45 mg/kg for 11 consecutive days, both compounds showed prolonged survival time (T/C = 142-145%), thus demonstrating efficacy against ras oncogene-containing tumors in vivo.

Neurofibromatosis type 2 and von Hippel-Lindau disease: from gene cloning to function.

Most of the genes for hereditary tumor syndromes cloned thus far have subsequently been shown to be mutated not only in the germlines and tumors from patients with the relatively rare inherited disease, but also in the much more common sporadic tumor counterparts in the general population. Thus, the isolation and functional characterization of genes associated with hereditary tumor syndromes have emerged as a major strategy to gain insights into some of the most fundamental mechanisms of tumorigenesis. The search for the genes causing two hereditary tumor syndromes of the nervous system, neurofibromatosis type 2 (NF2) and von Hippel-Lindau disease (VHL), has recently culminated in the cloning of both disease genes. This represents another successful application of the so-called positional cloning approach, i.e., the isolation of a hereditary disease gene with unknown function, based on the determination of its chromosomal location in the human genome. The gene for NF2, a syndrome typically associated with vestibular schwannomas and meningiomas, is homologous with a family of genes whose members appear to play an important role in bridging the cell membrane with the intracellular cytoskeleton, including moesin, ezrin, radixin, and protein 4.1. Recent mutation analyses have revealed that the NF2 tumor suppressor gene is frequently mutated not only in vestibular schwannomas and meningiomas from NF2 patients, but also in their sporadic counterparts, which represent approximately one-third of all human brain tumors. Furthermore, malignant human tumors seemingly unrelated to the NF2 syndrome, such as neural crest-derived malignant melanomas, as well as malignant mesotheliomas (a pleural mesoderm-derived tumor), have also been found to be frequently mutated or deleted in the NF2 locus, suggesting a broader role for the NF2 gene in the initiation and progression of human neoplasms. VHL is a rare tumor syndrome characterized by certain types of nervous system tumors (cerebellar and spinal hemangioblastomas as well as retinal angiomas), in conjunction with bilateral renal cell carcinomas and pheochromocytomas. Similar to NF2, recent genetic mutation studies have revealed that the VHL tumor suppressor gene is not only mutated in the hereditary tumors from VHL patients, but also in their sporadic counterparts. Importantly, the VHL gene represents the most frequently mutated cancer-related gene thus far identified in sporadic renal cell carcinoma. In contrast to most other hereditary cancer syndromes, however, VHL mutations are surprisingly specific for tumors typically associated with the VHL syndrome, and have not been detected in any other tumor type unrelated to VHL. The cloning and initial genetic characterization of the NF2 and VHL genes have now provided a rational basis for subsequent functional studies on the elucidation of the normal and tumor-associated cellular signaling pathways of these tumor suppressor genes.

Induction of the growth inhibitor IGF-binding protein 3 by p53.

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.

Farnesyltransferase inhibitors block the neurofibromatosis type I (NF1) malignant phenotype.

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.

Farnesyltransferase inhibitors are inhibitors of Ras but not R-Ras2/TC21, transformation.

Recent results from several laboratories including ours strongly suggest that farnesyltransferase (FT) inhibitors belonging to distinct chemical classes block growth of oncogenic Ras transformed cells at concentrations that do not affect the growth and viability of normal cells. This is despite blocking the farnesylation and thus the membrane association of Ras in both cell types. This is a paradox given the requirement for Ras function in normal cell growth. Recent evidence that R-Ras2/TC21 utilizes components of Ras signal transduction pathways to trigger cellular transformation (Graham et al., MCB 14, 4108-4115, 1994) prompted us to consider the possibility that R-Ras2/TC21 is involved in some aspects of the growth regulation of normal cells. If so, R-Ras2/TC21 may be compensating for Ras function in untransformed cells treated with FT inhibitors. In this study, we demonstrated that a cell active bisubstrate analog FT inhibitor, BMS-186511, completely blocked the function of oncogenic Ras, but did not affect the function of oncogenic R-Ras2/TC21, as determined by several criteria including inhibition of anchorage dependent and independent growth, reversal of transformed morphology and restoration of actin cytoskeleton. While it is known that TC21 protein becomes prenylated, it is not known whether it is farnesylated or geranylgeranylated. Our in vitro prenylation experiments indicate that R-Ras2/TC21 protein serves as a good substrate for FT as well as geranylgeranyltransferase I (GGTI) and thus provide the apparent molecular basis for these differences. Overall, these results, coupled with the ubiquitous expression of R-Ras2/TC21 in many cells including untransformed NIH3T3 cells, are consistent with the possibility that R-Ras2/TC21 may be one of the factors that render normal cells insensitive to the growth inhibitory action of FT inhibitors.

Regulation of the sequence-specific DNA binding function of p53 by protein kinase C and protein phosphatases.

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.

Cloning and characterization of a mouse gene with homology to the human von Hippel-Lindau disease tumor suppressor gene: implications for the potential organization of the human von Hippel-Lindau disease gene.

The human von Hippel-Lindau disease (VHL) gene has recently been identified and, based on the nucleotide sequence of a partial cDNA clone, has been predicted to encode a novel protein with as yet unknown functions [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. The length of the encoded protein and the characteristics of the cellular expressed protein are as yet unclear. Here we report the cloning and characterization of a mouse gene (mVHLh1) that is widely expressed in different mouse tissues and shares high homology with the human VHL gene. It predicts a protein 181 residues long (and/or 162 amino acids, considering a potential alternative start codon), which across a core region of approximately 140 residues displays a high degree of sequence identity (98%) to the predicted human VHL protein. High stringency DNA and RNA hybridization experiments and protein expression analyses indicate that this gene is the most highly VHL-related mouse gene, suggesting that it represents the mouse VHL gene homologue rather than a related gene sharing a conserved functional domain. These findings provide new insights into the potential organization of the VHL gene and nature of its encoded protein.

The neurofibromatosis 2 (NF2) tumour suppressor gene: implications beyond the hereditary tumour syndrome?

The cloning of the gene that causes neurofibromatosis type 2 (NF2), a hereditary tumour syndrome typically associated with brain tumours such as vestibular schwannomas and meningiomas, represents another successful application of the "positional cloning" approach--that is, the isolation of a hereditary disease gene of unknown function, based on the determination of its chromosomal location in the human genome. The NF2 gene is homologous to a family of genes whose products, including moesin, ezrin, radixin and protein 4.1, appear to have an important role in bridging the cell membrane and the intracellular cytoskeletion. Mutation analyses have revealed that the NF2 tumour suppressor gene is frequently mutated not only in vestibular schwannomas and meningiomas from NF2 patients, but also in their sporadic counterparts, which represent approximately one third of all human brain tumours. Furthermore, malignant human tumours seemingly unrelated to the NF2 syndrome, such as malignant melanomas (derived from the neural crest) and malignant mesotheliomas (derived from pleural mesoderm), also frequently have mutations or deletions at the NF2 locus, suggesting a broader role of the NF2 gene in the initiation and progression of human neoplasms.

Gene regulation by temperature-sensitive p53 mutants: identification of p53 response genes.

The ability of the p53 protein to act as a sequence-specific transcriptional activator suggests that genes induced by p53 may encode critical mediators of p53 tumor suppression. Using a tetracycline-regulated p53 expression system and cDNA library subtraction procedure, we identified several p53-induced gene transcripts in human Saos-2 osteosarcoma cells that are novel on the basis of their size, regulation, and low abundance. Wild-type p53-dependent induction of these transcripts was observed in cells that are growth arrested by p53, as well as in cells that undergo apoptosis upon expression of an inducible wild-type p53 transgene. These results show that p53 activates the expression of numerous response genes and suggest that multiple effectors may play a role in mediating cellular functions of p53.

Genetic basis of neurological tumours.

Neurological tumours are common neoplasms of both adults and children. Recent studies have begun to delineate the genetic abnormalities that underlie such tumours, and have implicated two classes of genes, oncogenes and tumour suppressor genes. Most investigations have focused on those astrocytomas that affect the cerebral hemispheres of adults, since these are the most common and malignant brain tumours. The high-grade astrocytomas that affect adults, such as glioblastoma multiforme, often have amplification of the epidermal growth factor receptor (EGFR) oncogene and loss of a variety of chromosomal loci that probably harbour tumour suppressor genes. Of the various tumour suppressor gene loci, the p53 gene on chromosome 17p has been studied most closely and has been shown to be mutated in both low- and high-grade astrocytomas. These genetic alterations may provide a means for subdividing astrocytomas into diagnostic categories. For instance, p53 gene mutations occur more commonly in glioblastomas from young adults and women, while EGFR gene amplification is more common in glioblastomas from older adults and men. For the other primary CNS tumours, genetic studies remain in their infancy. The neurocutaneous syndromes, such as neurofibromatosis types 1 and 2, have provided unique insights into neurological oncogenesis. The NF1 gene on chromosomes 17q and its product, neurofibromin, may be important in the formation of neurofibrosarcomas, while the NF2 gene on chromosome 22q and its product, merlin, are probably involved in the formation of schwannomas and other nervous system tumours. The further characterization of these and other neurological tumour genes will undoubtedly illuminate many other areas in neurooncology.

Frequent NF2 gene transcript mutations in sporadic meningiomas and vestibular schwannomas.

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.

P-glycoprotein, HER-2/neu, and mutant p53 expression in human gynecologic tumors.

BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types.

The neurofibromatosis 2 gene (NF2) has recently been isolated and predicted to encode a novel protein related to the moesin-ezrin-radixin family of cytoskeleton-associated proteins. Here we describe a novel isoform of the NF2 transcript that shows differential tissue expression and encodes a modified C terminus of the predicted protein. Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumour types including melanoma and breast carcinoma. These findings provide evidence that alterations in the NF2 transcript occur not only in the hereditary brain neoplasms typically associated with NF2, but also as somatic mutations in their sporadic counterparts and in seemingly unrelated tumour types. The NF2 gene may thus constitute a tumour suppressor gene of more general importance in tumorigenesis.

Molecular cloning and characterization of alternatively spliced transcripts of the mouse neurofibromatosis 2 gene.

The human neurofibromatosis 2 (NF2) gene has recently been isolated and predicted to encode a novel protein named merlin. Based on its high homology to the moesin-ezrin-radixin family of proteins, it may be involved in mediating interactions between the plasma membrane and the cytoskeleton. Here we report the isolation and characterization of multiple transcript isoforms of the mouse NF2 gene. The full length coding complementary DNA sequence of transcript isoform I is 1788 base pairs in length, shares 90% sequence identity with the human NF2 complementary DNA, and encodes a putative protein of 596 amino acids sharing 98% homology with the human merlin protein. Transcript isoforms II and III carry a 45- and 16-base pair insertion, respectively, at nucleotide 1740 at the 3' end, generated by two different modes of alternative splicing; both insertions introduce premature termination codons. Thus, transcript isoforms II and III predict proteins of 591 and 584 amino acids with altered COOH-termini of more hydrophilic character as compared to isoform I. Northern blot analysis and reverse transcription-polymerase chain reaction analysis indicate that the mouse NF2 gene is widely expressed in different tissue types and that the alternative transcripts are variantly expressed. The results presented here indicate high conservation of the NF2 gene during evolution and suggest a possible role for the COOH-terminus in mouse merlin function.

Identification of p53 mutations by means of single strand conformation polymorphism analysis in gynaecological tumours: comparison with the results of immunohistochemistry.

The tumour-suppressing gene p53 may undergo mutation by a variety of mechanisms, thus losing its tumour-suppressing activity, and ultimately behaving like an oncogene. The PAb 1801 monoclonal antibody is known to recognise both wild type and mutated p53, although in practice it seems to show a higher reactivity with the mutated gene product in several human tumours. We studied p53 overexpression in a series of 36 human tumours (17 mammary ductal infiltrating carcinomas, 11 endometrial carcinomas and 8 uterine cervical carcinomas) by means of immunohistochemistry using the PAb 1801 antibody and the streptavidin-biotin peroxidase technique. Furthermore, all tumours were screened for mutations in the "hot spot" regions of the p53 gene (exons 5 to 8) by means of SSCP (single strand conformation polymorphism) DNA analysis following amplification of the target exons using the polymerase chain reaction. A good correlation (75-100%) between positive immunohistochemistry and p53 mutations was observed in mammary and endometrial cancer, whereas mutations were detected in only two out of seven immunoreactive cervical carcinomas. Following these results, immunohistochemistry with the PAb monoclonal antibody may be safely used as a screening tool for the detection of mutated p53 in clinical samples of mammary and endometrial cancer, whereas it should be complemented with DNA analysis in cervix carcinoma.