Point mutational inactivation of the retinoblastoma antioncogene.

The retinoblastoma (Rb) antioncogene encodes a nuclear phosphoprotein, p105-Rb, that forms protein complexes with the adenovirus E1A and SV40 large T oncoproteins. A novel, aberrant Rb protein detected in J82 bladder carcinoma cells was not able to form a complex with E1A and was less stable than p105-Rb. By means of a rapid method for the detection of mutations in Rb mRNA, this defective Rb protein was observed to result from a single point mutation within a splice acceptor sequence in J82 genomic DNA. This mutation eliminates a single exon and 35 amino acids from its encoded protein product.

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.

Frequent p53 gene mutations in blast crisis of chronic myelogenous leukemia, especially in myeloid crisis harboring loss of a chromosome 17p.

We investigated chromosome alterations and mutations of the p53 gene in 118 samples from 92 patients with chronic myelogenous leukemia in various clinical phases, i.e., chronic phase, accelerated phase, and blast crisis (BC). Single-strand conformation polymorphism analysis and subsequent nucleotide sequencing disclosed no alteration of the p53 gene in chronic phase (no mutation in 80 samples), while five of 31 BC samples showed point mutations: four in myeloid and one in lymphoid crisis. One of seven accelerated phase samples also showed a p53 gene mutation. Ten of 31 BC samples showed loss of one of the short arms of chromosome 17 (17p) through the formation of isochromosome 17q, i(17q), or unbalanced translocations. Loss of heterozygosity at the p53 locus in the accelerated phase and BC was detected only in two cases with i(17q) but not in seven cases with normal chromosome 17 homologues, suggesting that loss of one p53 allele is rare without cytogenetically detectable loss of a 17p. Among those six samples with p53 gene mutations, five showed loss of a 17p cytogenetically, and only one lymphoid crisis case exhibited normal chromosome 17 homologues. Thus, mutations of the p53 gene were closely associated with myeloid crisis with loss of a 17p (four mutations in ten samples), in contrast to myeloid crisis with normal chromosome 17 homologues (zero in 13) or lymphoid crisis (one in seven). Our results also suggest that alterations of the p53 gene might occur after loss of a 17p during the course of chronic myelogenous leukemia.

Role and mutational heterogeneity of the p53 gene in hepatocellular carcinoma.

The mutational spectrum of the p53 gene was analyzed in 53 hepatocellular carcinomas. Somatic mutations of the p53 gene were detected in 17 cases (32%). Among these 17 mutations, 9 were missense mutations; the mutations in the other 8 cases were nonsense mutations, deletions, or mutations at the intron-exon junctions. These mutations were found in a wide region stretching from exon 4 to exon 10 without any single mutational hot spot. G:C to T:A transversions were predominant, suggesting the involvement of environmental mutagens in the mutagenesis of the p53 gene in a subset of the hepatocellular carcinoma cases. Mutations of the p53 gene occurred frequently in advanced tumors, although several tumors in the early stages also showed mutations. A deletion map of chromosome 17 was constructed by using 10 polymorphic probes and was compared with the p53 gene mutation in each case. Loss of heterozygosity (LOH) on chromosome 17p was observed in 49% of the cases (24 of 49), and two commonly deleted regions were detected (around the p53 locus and at 17p13.3 to the telomere). Sixteen of the 17 cases with p53 gene mutations showed LOH around the p53 locus, and mutations were rare in hepatocellular carcinomas without LOH. However, no mutations were detected in 8 cases with LOH on 17p, suggesting the possibility that an unidentified tumor suppressor gene(s) located on 17p may have also been involved in hepatocarcinogenesis.

p53 gene mutations and abnormal retinoblastoma protein in radiation-induced human sarcomas.

The potentially carcinogenic effect of therapeutic irradiation has been recognized for many years. Second malignancies, usually sarcomas, are known to arise within or at the edge of radiation fields after a period of several years after the initial radiation exposure. We analyzed tumor cells derived from seven radiation-induced tumors for abnormalities in tumor suppressor genes p53 and retinoblastoma at the DNA sequence and/or protein level. p53 mutations were detected by exon-specific polymerase chain reaction amplification and single-strand conformation polymorphism analysis of exons 5-8 followed by direct genomic sequencing of those tumors exhibiting a variant pattern. The p53 gene was abnormal in three of six sarcomas studied. Retinoblastoma gene analysis was performed by Western immunoblot; retinoblastoma protein was under-phosphorylated in three of seven tumors and absent in one other. In all, six of seven radiation-induced human tumors have abnormalities of one or both suppressor genes. Inactivation of tumor suppressor genes by ionizing radiation may contribute to radiation carcinogenesis.

Altered p53 status correlates with differences in sensitivity to radiation-induced mutation and apoptosis in two closely related human lymphoblast lines.

Previous work identified TK6 and WTK1 as human lymphoblast cell lines from one donor that have different capacities to catalyze recombination and that vary significantly in their response to ionizing radiation. WTK1 cells are more resistant to the toxic effects of X-rays yet more sensitive to induced mutation. We demonstrate here that although both cell lines contain equal levels of p53 mRNA, baseline protein levels are 4 times higher in WTK1. Irradiation leads to higher levels of p53 protein in both lines but to a greater extent in TK6. TK6 contains a wild-type p53 sequence, while WTK1 has a homozygous mutation in codon 237 of exon 7. We also observed a significant difference in the kinetics but not the overall degree of apoptosis induced by X-rays in these cells; apoptotic death is delayed for 3 days in WTK1. We hypothesize that this p53 mutation is responsible for the difference in apoptosis as well as for the differences in mutability and mutational spectra reported previously.

Inactivation of the retinoblastoma gene in human bladder and renal cell carcinomas.

The retinoblastoma (RB) gene was the first tumor suppressor gene isolated and its inactivation is associated with the pathogenesis of several types of human cancer. In this study, we investigated the involvement of the RB gene in bladder and renal cell carcinomas by determining the loss of heterozygosity (LOH) at the RB locus and by DNA, RNA, and protein analysis of the RB gene. Whenever possible, the latter included Western blotting and immunohistochemical staining of the RB protein. In bladder carcinoma, 2 of the 8 cell lines we studied had an inactivated RB gene; one cell line lacked RB expression without a gross RB deletion, whereas the other cell line expressed only the underphosphorylated form of the RB protein. None of 16 low-grade noninvasive bladder carcinomas showed an alteration in RB protein by direct Western blot analysis, whereas 2 of 14 high-grade, invasive tumors had no RB protein as measured by both Western blotting and immunohistochemical staining. This suggests that the loss of RB function may be more important in the progression of bladder cancer than in its initiation, although more extensive studies are required. LOH within the RB locus was observed in 5 of 27 informative cases of primary bladder, ureter, or renal pelvis carcinoma. However, none of the 5 cases with LOH at the RB locus had a functional loss of RB protein expression. In renal cell carcinoma, one of the 12 cell lines had a gross homozygous deletion of the RB gene, and 2 of 32 primary tumors were negative for RB protein expression. LOH at the RB locus also was found in only 2 of 30 informative cases, one of which lacked RB expression. These results are the first to demonstrate the involvement of RB inactivation in the development of advanced primary bladder carcinoma and suggest that RB loss could have a role in certain renal cell carcinomas. Our data, however, show no correlation between LOH at the RB locus in bladder cancer and actual inactivation of the RB gene at the protein level. This may suggest that there is a second tumor suppressor or recessive cancer gene on chromosome 13 in bladder cancer and/or that the mechanism of RB inactivation in bladder cancer frequently involves independent mutations of each RB allele.

Accumulation of wild type p53 protein in human astrocytomas.

We have previously described 10 astrocytomas with accumulation of p53 protein but no mutations in p53 exons 5-8, and we have suggested that they might represent overexpression of wild type protein or mutations in less conserved regions of the gene. To investigate these possibilities further, we studied the tumors with immunohistochemistry for wild type and mutant p53 protein and showed that all cases stained with the wild type PAb 1801 antibody but only one case stained with the mutant-specific PAb 240 antibody. To support the hypothesis that the accumulated p53 protein is wild type in most cases, we used single-strand conformation polymorphism analysis and DNA sequencing to evaluate p53 exons 4, 9, and 10 and did not detect mutations at these loci. Although the product of the MDM2 oncogene binds wild type p53 and may account for p53 accumulation, slot-blot analysis of these astrocytomas did not detect MDM2 gene amplification. Thus, evidence suggests that some astrocytomas may accumulate wild type p53 protein but not as a result of MDM2 gene amplification. Furthermore, PAb 1801 immunohistochemistry may not be an adequate method of screening astrocytomas for p53 mutations.

Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas.

We analyzed mutations of the p53 gene by single-strand conformation polymorphism analysis of polymerase chain reaction products and direct sequencing through all coding exons and exon-intron junctions in 32 cases with esophageal squamous cell carcinoma. Mutations were detected in 15 of 32 (47%) tumor samples, in which G:C to T:A transversions were rather frequent (33%). Previously, we reported deletion of chromosome 17p where the p53 gene is located in 45% of Japanese esophageal squamous cell carcinoma, and here the relationship between mutation of the p53 gene and loss of 17p was analyzed. Mutations were observed in 12 of 16 patients with loss of 17p, whereas only 2 of 11 without loss were positive for mutations, suggesting that mutations of the p53 gene were closely associated with a 17p deletion. Furthermore, we immunohistochemically analyzed the expression of p53 protein in esophageal squamous cell carcinoma tumor tissues using a monoclonal antibody. Five of 6 tumors with missense mutations of the p53 gene were positively stained, while in tumors with nonsense mutations or without mutations of the p53 gene staining was very weak or negative. These results suggest a good correlation between mutations and abnormal expression of the p53 gene.

Characterization of intragenic deletions in two sporadic germinal mutation cases of retinoblastoma resulting in abnormal gene expression.

Two gross intragenic deletions of the retinoblastoma (RB) gene from patients with sporadic hereditary disease were analysed in detail. This study was undertaken to determine whether there were common mechanisms for these deletions and whether changes found in the tumors were identical to those found in the constitutional cells. Short repeats at the deletion breakpoints were found in both tumors, one resulting in a 2.0 kb deletion including exons 21 and 22, and the other producing a 3.7 kb deletion including exons 14-17. In addition, the identical sequence changes documented in genomic DNAs and transcripts of these tumors were also observed in the constitutional cells. Both deletions were in-frame and resulted in a truncated transcript and protein identical to the expected size based on the exons deleted. These studies provide further documentation of the events which represent the actual 'two hits' originally hypothesized to be responsible for retinoblastoma development.

Single strand conformation polymorphism analysis of androgen receptor gene mutations in patients with androgen insensitivity syndromes: application for diagnosis, genetic counseling, and therapy.

Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes, a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males. In this report, we address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis, genetic counseling, and molecular subclassification of affected patients and their families. DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied. Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing. Female family members were also studied to identify heterozygote carriers. Point mutations in the AR gene were identified in all six patients, and all mutations caused amino acid substitutions. One patient with incomplete androgen insensitivity was a mosaic for the mutation. Four of the five mothers, as well as a young sister of one patient, were carriers of the mutation present in the affected child. Our data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome. Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy, and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance. The identification of carriers is of substantial clinical importance for genetic counseling.

Structure and partial genomic sequence of the human retinoblastoma susceptibility gene.

This report describes the genomic organization of the human retinoblastoma susceptibility locus. This gene spans approximately 200 kb of DNA within human chromosome 13, band q14. The previously determined cDNA sequence comprises 27 exons, ranging in size from 31 bp to 1873 bp, and 26 introns, ranging in size from 80 bp to 70,500 bp. We have mapped the positions of the exons and the positions of the recognition sites for six restriction endonucleases. We also present the sequence of 9.2% of the locus (18,335 bp), including approximately 200 bp of intron sequence immediately flanking each exon. This map of a wild-type allele will form the foundation for future studies of mutant, oncogenic alleles at this locus.

Loss of heterozygosity on chromosome 17 and mutation of the p53 gene in retinoblastoma.

Loss of heterozygosity (LOH) on chromosome 17 and mutations of the p53 gene were examined in 25 retinoblastomas (RB), consisting of three familial tumors, nine hereditary tumors without family history, 11 non-hereditary tumors, one recurrent tumor and one lung-metastatic tumor. LOH on chromosome 17 was detected in only one of the 23 primary RB. No mutations of the p53 gene were detected in the primary tumors. A recurrent tumor showed LOH on the short arm region of chromosome 17. LOH on chromosome 17 and a point mutation of the p53 gene were also detected in a metastatic tumor. These results suggest that LOH on chromosome 17 and mutation of the p53 gene may not be associated with the development of primary RB, but may play a role in the progression of RB.

p53 gene analysis of ovarian borderline tumors and stage I carcinomas.

Mutations of the p53 gene are common in human ovarian carcinomas; however, their role in the early development of ovarian cancer is unclear. Twelve ovarian borderline tumors (BTs; eight of them p53 immunopositive) and 10 stage I carcinomas (four of them p53 immunopositive) were studied for genetic alterations in the p53 gene. The study was based on single-strand conformation polymorphism (SSCP) analysis and DNA sequencing of exons 2 through 11 of the p53 gene using DNA preparations from microdissected tumors. Mutations were found in 40% of the carcinomas (including a borderline component adjacent to carcinoma in one lesion) but in none of the pure BTs. These findings suggest that p53 mutations may not be commonly associated with the borderline phenotype of ovarian epithelial tumors but may occur during malignant transformation.

p53 expression in ovarian borderline tumors and stage I carcinomas.

Seventy-nine ovarian serous and mucinous borderline tumors, 36 stage I carcinomas and 39 stage II-IV carcinomas were studied for p53 protein accumulation with monoclonal antibody PAb1801.p53 protein was expressed in 14% of borderline tumors, 36% of stage I carcinomas, and 64% of higher stage carcinomas. All immunopositive carcinomas accumulated p53 protein in the primary tumor, and 95% of them showed concordance in staining among different tissue blocks. A difference in frequency of p53 protein accumulation between stage I and higher stage serous carcinomas was not statistically significant. p53 positivity was associated with microinvasion, microcarcinoma and coexistent carcinoma in mucinous borderline tumors (P = .025). An association between p53 protein expression and poor tumor differentiation in Stage I carcinomas as statistically significant (P = .03). p53 positivity was observed in a poorly differentiated endometrioid carcinoma as well as in adjacent benign endometriotic tissue. These results suggest that p53 abnormalities may be early events in ovarian cancer, possibly contributing to malignant transformation of some borderline tumors, endometriosis and other carcinoma precursors.

Frequency and spectrum of p53 mutations in gastric cancer--a molecular genetic and immunohistochemical study.

The p53 tumour-suppressor gene plays an important role in gastric carcinogenesis. In an analysis of the spectrum of mutations of the p53 gene seen in 56 primary gastric carcinomas of various types and grades of differentiation, the entire coding sequence (exons 2-11) of the p53 gene was screened by single-strand conformation polymorphism analysis and direct genomic sequencing of polymerase chain reaction products. Intragenic restriction site polymorphisms and the probe YNZ22 were used for the detection of loss of heterozygosity (LOH) of the p53 gene locus on chromosome 17p. p53 overexpression was studied with the anti-p53 antibody CM-1. A total of 21 somatic alterations of the p53 gene were found. Twenty were base-pair substitutions, and one was an eight base-pair deletion. Six tumours with p53 mutations revealed LOH. Abnormalities in p53 expression were found in 17 tumour samples, of which 16 had gene mutations. The spectrum of mutations observed was consistent with the predicted spectrum for dietary mutagens associated with the metabolism of nitrogenous compounds, resulting in deamination of nucleic acids. Our findings suggest that p53 could be a primary target for mutations associated with dietary carcinogens in gastric carcinogenesis.

Rapid detection of loss of heterozygosity of chromosome 17p by polymerase chain reaction-based variable number of tandem repeat analysis and detection of single-strand conformation polymorphism of intragenic p53 polymorphisms.

Intragenic restriction site polymorphisms in amino acid residue 72 in exon 4 and a Mspl polymorphism in intron 6 of the p53 tumour suppressor gene can both serve as polymorphic markers. Probe YNZ22 (D17S5) is a highly polymorphic, variable number of tandem repeat (VNTR) marker which maps to chromosome 17p13.1 where the p53 gene is located. Locus specific amplification by polymerase chain reaction (PCR) technique and subsequent non-isotopic single-strand conformation polymorphism analysis of the PCR fragments was used for the detection of loss heterozygosity (LOH) of 17p including the p53 gene locus. In combination with a PCR-based method for the analysis of the VNTR locus D17S5 using unique sequences flanking the polymorphic region of YNZ22 we investigated tumour DNA and corresponding constitutional DNA from 69 patients, including 39 patients with gastric cancer, 21 patients with osteosarcomas and 9 patients with Ewing's sarcomas. Using all three methods, 49/69 (71%) patients were informative for LOH, which revealed allelic loss in 5/39 (12.8%) gastric cancers, 1/9 (11.1%) Ewing's sarcoma, and 4/20 (20%) osteosarcomas.

Immature teratomas of different origin carried by a pregnant mother and her fetus.

We describe the case of a pregnant mother and her fetus who both carried teratomas during the pregnancy. The fetus was diagnosed at 38 weeks' gestation to have an intracranial mass, which was later determined to be an immature teratoma. During a cesarean section delivery, an ovarian tumor was found in the 27-year-old mother that was also diagnosed to be an immature teratoma. Because of the similar histology of the tumors carried by both mother and child, a single clonal origin was suspected. Using polymerase chain reaction (PCR) and electrophoresis of highly polymorphic DNA satellite sequences, we determined that the origin of the intracranial teratoma carried by the child was independent of the mother's tumor. We also examined the p53 tumor suppressor gene in constitutional cells from both mother and child for the possible presence of a cancer-predisposing inherited mutation, but none was found. To our knowledge, this is the first report of the simultaneous occurrence of independent malignant immature teratomas in a mother and child during pregnancy.

Chromosome 14 marker appearance in a human B lymphoblastoid cell line of nonmalignant origin.

A human B cell lymphoblastoid cell line of nonmalignant origin, derived 16 years ago, now carries a 14q+ marker chromosome in 100% of the cells analyzed. This marker was not apparent in karyotypes prepared 1 year after establishment of the line and, thus, presumably arose in culture. The cell line now grows more rapidly than typical B cell cultures and has an unusually high cloning efficiency (60%-90%). Using Southern blot hybridization, we have analyzed the arrangement of 13 onc genes that are often associated with human malignancy, including c-myc. None of these onc genes appear to be rearranged in this cell line.

Cytogenetic and molecular studies of a familial renal cell carcinoma.

In a previously studied family with inherited renal cell carcinoma (RCC), RCC was shown to segregate with a constitutional balanced t(3;8)(p14.2;q24.1). In addition, we recently showed that in a RCC tumor from this family the constitutional translocation became unbalanced, suggesting a genetic mechanism that may be associated with the primary genetic events of tumorigenesis. We now report that the RCC tumor cells from this case showed additional cytogenetic alterations, possibly related to tumor progression, which include an additional tumor-specific translocation involving band 14 of chromosome 13. Because this band contains the retinoblastoma (RB) gene, we examined the tumor for aberrations in the RB gene using DNA sequence polymorphism analysis and pulsed-field gel electrophoresis (PFGE), but did not detect alterations in the RB gene.