Fibronectin synthesis by human tubular epithelial cells in culture: effects of PDGF and TGF-beta on synthesis and splicing

Mutations of p53 tumor suppressor gene are the most common genetic alterations in a variety of human carcinomas. The sites of p53 mutations, however, vary in different cancers. The present study was designed to characterize p53 mutations in 40 primary human renal cancer specimens using hot-start-PCR-single-strand conformation polymorphism (SSCP) analysis, sequencing of PCR product and immunohistochemistry. DNA extracted from microdissected paraffin-embedded sections was amplified by hot-start-PCR using oligonucleotide primers specific for exons 4-9 of p53. The mutations were analyzed by PCR-SSCP technique and the generated fragments were denatured and analyzed by 6% polyacrylamide gel electrophoresis. The samples showing a band shift were denatured and sequenced using the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, Cleveland, Ohio). Genomic DNA from control samples containing wild-type p53 alleles was sequenced in parallel for confirming mutations in samples that were positive for p53 in the PCR-SSCP analysis. The results of these experiments demonstrate that: (1) there were mutations in p53 exon 5 and 8 in 35% (14 out of 40 samples) of human renal cancer tissues as revealed by PCR-SSCP analysis; (2) DNA sequencing of samples showing frame-shift have hot spot of p53 mutation on exon 8 at codon 244 (GGC-->TGC) and exon 5 at codon 132 [AAG (Lys)-->AGG (Arg)]. This mutation in p53 exon 5 at codon 132 is novel and has not yet been reported; (3) immunohistochemical staining of p53 in renal cancer tissue using mouse anti-human p53 monoclonal antibody, clone PAb 1801, correlated with the p53 mutation assessed by PCR-SSCP. No correlation was found between p53 mutations and tumor stage and grade of renal cancer.     

Molecular characterization of germline mutations in neurofibromatosis 2 in two families

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder that predisposes patients to central nervous system tumors. It is caused by mutations in the NF2 tumor suppressor gene, which is located on chromosome 22q12. We studied 2 multigenerational NF2 families (three members of family 1 and the proband of the family) by gene mutation analysis and clinical assessment. One member of family 1 had a 169 C-->T point mutation at codon 57 of exon 2 and had a severe phenotype. His father had a silent 1113 C-->T point mutation at codon 371 of exon 11 and had a normal phenotype. The proband of family 2 had a deletion at nucleotide 720 G (codon 240) of exon 8. This led to a frameshift and termination at codon 250, and a severe NF2 phenotype. Our results indicate that clinical abnormalities can be present in carriers. Nonsense and frameshift mutations in the NF2 tumor suppressor gene are associated with phenotypes. The clinical abnormalities can develop at a young age.     

Molecular basis of hereditary fructose intolerance in Italy: identification of two novel mutations in the aldolase B gene

We screened the aldolase B gene in 14 unrelated Italian patients with hereditary fructose intolerance (HFI), and found two novel disease related mutations: a single nucleotide deletion in exon 2 (delta A20) that leads to an early stop codon, and a C-->T transition in exon 8 that substitutes an Arg with a Trp residue at codon 303 (R303W).     

Fas-mediated apoptosis with normal expression of bcl-2 and p53 in lymphocytes from aplastic anaemia

The p53 tumor suppressor gene is critical in regulating cell proliferation following DNA damage, and disruption of p53 protein function by mutation has been implicated as a factor responsible for resistance of tumor cells to chemotherapeutic agents. Our studies were initiated by asking whether the translational product of the p53 gene is associated with cisplatin resistance in the 2780CP human ovarian tumor model. We have demonstrated by single-strand conformation polymorphism analysis and sequencing that p53 in parental cisplatin-sensitive A2780 cells was wild type. In 2780CP cells, however, a mutation was found in exon 5 at codon 172 (Val to Phe). Interestingly, exposure to X-rays resulted in p53 induction in both A2780 and 2780CP tumor models. The p53 increases by the ionizing radiation were accompanied by concomitant increases in levels of the p53-regulated p21Waf1/Cip1 protein and led to arrest of cells in G1 phase of the cell cycle. A yeast functional assay confirmed that p53 in A2780 was wild type, but, more importantly, it provided evidence that the p53 mutation in 2780CP cells was temperature sensitive and heterozygous. These experiments demonstrate that sensitive and resistant cells have normal p53 functions, despite the presence of p53 mutation in the 2780CP model. In parallel investigations using the Western technique, exposure of A2780 cells to clinically relevant concentrations of cisplatin (1-20 microM) resulted in time- and dose-dependent increases in p53, together with coordinate increases in p21Waf1/Cip1. In contrast, cisplatin did not induce these proteins in 2780CP cells to any significant degree. The results indicate that a defect exists in the signal transduction pathway for p53 induction following cisplatin-induced DNA damage in 2780CP cells, and this may represent a significant mechanism of cisplatin resistance. Furthermore, induction of p53 in 2780CP cells by X-rays, but not cisplatin, strongly suggests that independent pathways are involved in p53 regulation for the two DNA-damaging agents.     

Chronic ultraviolet exposure-induced p53 gene alterations in Sencar mouse skin carcinogenesis model

Alterations of the tumor suppresser gene p53 have been found in ultraviolet radiation (UVR) related human skin cancers and in UVR-induced murine skin tumors. However, links between p53 gene alterations and the stages of carcinogenesis induced by UVR have not been clearly defined. We established a chronic UVR exposure-induced Sencar mouse skin carcinogenesis model to determine the frequency of p53 gene alterations in different stages of carcinogenesis, including UV-exposed skin, papillomas, squamous-cell carcinomas (SCCs), and malignant spindle-cell tumors (SCTs). A high incidence of SCCs and SCTs were found in this model. Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. DNA was isolated from 40 paraffin-embedded normal skin, UV-exposed skin, and tumor sections. The p53 gene (exons 5 and 6) was amplified from the sections by using nested polymerase chain reaction (PCR). Subsequent single-strand conformation polymorphism (SSCP) assay and sequencing analysis revealed one point mutation in exon 6 (coden 193, C-->A transition) from a UV-exposed skin sample, and seven point mutations in exon 5 (codens 146, 158, 150, 165, and 161, three C-->T, two C-->A, one C-->G, and one A-->T transition, respectively) from four SCTs, two SCCs and one UV-exposed skin sample. These experimental results demonstrate that alterations in the p53 gene are frequent events in chronic UV exposure-induced SCCs and later stage SCTs in Sencar mouse skin.     

Combining stimulus fading, reinforcement, and extinction to treat food refusal

The expression of mismatch repair proteins hMSH2 and hMLH1 was investigated in human ovarian cancer cell lines and in biopsies of ovarian carcinomas obtained from 20 patients undergoing surgical operation. By Western blotting analysis hMSH2 protein was detected in all the tumor samples analyzed and in eight out of nine human ovarian cancer cell lines, while hMLH1 was undetectable in four out of 20 ovarian tumors and in five out of nine human ovarian cancer cell lines analyzed. The possible presence of frameshift mutations in the BAX gene, which contains a sequence of eight contiguous guanines in its third exon, was tested in all the samples. All the cell lines presented the normal alleles for the BAX gene while only in one of the tumor samples a heterozygous frameshift mutation was found. The frameshift mutation was associated to a low, almost undetectable, level of BAX protein which was instead present at much higher levels in all the other samples investigated. The results indicate that frameshift mutations in the BAX gene, possibly arising as a consequence of microsatellite instability (detectable in these tumors), is detectable in human ovarian cancer although quantitatively it does not appear to be a major determinant of the low apoptotic response to chemotherapy observed in ovarian cancer cells.     

Association between human cancer and two polymorphisms occurring together in the p21Waf1/Cip1 cyclin-dependent kinase inhibitor gene

BACKGROUND: The cyclin-dependent kinase inhibitor gene p21Waf1/Cip1 plays a role in signaling cellular growth arrest. In response to DNA damage, p21 is induced by the p53 gene, thereby playing a direct role in mediating p53-induced G1 arrest. Alterations in this gene may adversely affect regulation of cellular proliferation and increase susceptibility for cancer. Two polymorphisms have previously been characterized in the p21 gene: a C-->A transversion at codon 31 (ser-->arg) and a C-->T transition 20 nucleotides downstream from the 3' end of exon 3. METHODS: The codon 31 polymorphism in exon 2 of the p21 gene was identified by restriction digestion (Alw26I) of products amplified by polymerase chain reaction (PCR). The polymorphism downstream from exon 3 of the p21 gene was identified by single strand conformation polymorphism (SSCP) analysis of PCR amplified products and was confirmed by PstI enzyme restriction digestion. DNA variant alleles were confirmed by direct DNA sequencing. The entire coding region and the promoter region (p53 binding domain) of the p21 gene were screened for mutations by SSCP analysis or DNA sequencing. RESULTS: The two polymorphisms were found in 18 of 96 tumor samples lacking p53 alterations (18.8%). Nine of 54 prostate adenocarcinoma samples (16.7%) contained both p21 variants, whereas 9 of 42 squamous cell carcinomas of the head and neck (21.4%) displayed both polymorphisms. Of the 110 controls examined, 10 (9.1%) had both alterations. Both p21 polymorphisms occurred together in all samples examined and there was no indication of mutation in the coding region of the p21 gene or in the p53 binding domain of the promoter region. CONCLUSIONS: These data suggest that p21 gene variants may play a role in increased susceptibility for the development of some types of cancer. In the current study, the authors demonstrated that the occurrence of these two polymorphisms is increased in prostate adenocarcinoma and squamous cell carcinoma of the head and neck. The polymorphic sites may be directly responsible for this apparent increased susceptibility or they may be linked to regulatory region alterations.     

Mutational analysis of the BRCA1 tumor suppressor gene in endometrial carcinoma

OBJECTIVE: To investigate whether alteration of BRCA1 tumor suppressor gene occurs in sporadic endometrial carcinomas. METHODS: Genomic DNAs were prepared from 33 consecutively collected endometrial carcinoma tissues for BRCA1 mutational analysis. To screen for BRCA1 mutation, polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) technique were used with 41 overlapping PCR primer pairs for the 23 coding exons of BRCA1. Tumors that demonstrated SSCP variants were further subjected to direct DNA sequencing in the appropriate exons to identify the DNA alteration. RESULTS: In addition to detecting a previously described polymorphism in exon 11, single strand conformation polymorphism analysis of the 33 endometrial cancers identified 3 tumors with mobility shifts. Two tumors shifted in exon 3 and showed the same pattern of band shift. The other tumor shifted in exon 9. DNA sequencing revealed sequence alterations in the 3 tumors; all appeared heterozygous. In the 2 tumors shifted in exon 3, the sequence alteration caused no amino acid change and was consistent with an infrequent silent polymorphism. In the third tumor, a missense alteration at codon 191 was detected and was recognized as germline in origin. CONCLUSIONS: Because a normal allele of BRCA1 was retained in the tumor where a germline missense alteration was detected, the heterozygous DNA alteration should not be cancer predisposing in terms of the two-hit model for inactivation of the tumor suppressor gene. We conclude that mutation of BRCA1 may not be involved in the development of sporadic endometrial cancer.     

4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-3-deoxy-D-manno-2-octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus

A thyroglobulin (Tg) synthesis defect in Dutch goats causes congenital goiter and hypothyroidism. The disease is inherited in an autosomal recessive way and is linked to restriction fragment length polymorphisms (RFLPs) in the Tg gene. Previous studies showed that Tg mRNA isolated from the goiters was of normal size (8.4 kilobases). Translation of high mol wt polysomal Tg mRNA isolated from goiter in a cell-free rabbit reticulocyte lysate resulted in a single 35,000 mol wt Tg polypeptide. Tg antigens analyzed in T4-arrested goiters were glycosylated and had mol wt of 40,000 and 32,000. The aim of this study was to identify the molecular lesion responsible for this disease. Polysomal Tg mRNA, therefore, was isolated, and cDNA was made using oligonucleotides as primers. This cDNA was multiplied by the polymerase chain reaction and cloned. In comparing the normal and abnormal sequences, we found a C-->G point mutation in exon 8 causing a change from TAC (Tyr)-->TAG (termination signal) at amino acid position 296. This mutation resulted in the appearance of a KpnI restriction site in the goiter DNA. The sequence of Tg mRNA preceding the stop codon was equal for normal and goitrous goats, except for one C-->T mutation in exon 5 which gave a Ser-->Leu transition. The KpnI site introduced by the C-->G point mutation was present in chromosomal DNA of the goitrous goats, making it possible to distinguish goats heterozygous for the defect from normal and goitrous animals. We calculated that the stop codon in exon 8 would result in a Tg polypeptide chain with a mol wt of 39,000, in good agreement with the mol wt of the in vitro and in vivo translation products. In conclusion, the C-->G mutation causing a stop codon in exon 8 is responsible for the Tg synthesis defect in Dutch goats.     

Transforming growth factor-beta receptors on human endometrial cells: identification of the type I, II, and III receptors and glycosyl-phosphatidylinositol anchored TGF-beta binding proteins

BACKGROUND: Mutation of the p53 tumor suppressor gene is the most commonly found genetic alteration in human cancer. The E6 gene product of human papillomavirus (HPV) 16 and 18 can inactivate the p53 protein by promoting its degradation. Because most HPV-positive cervical carcinoma cell lines contain wild-type p53 whereas HPV-negative cell lines have point mutations in the p53 gene, a major role in the development of HPV-negative cervical cancer has been attributed to p53. Recent studies, however, have observed no consistent presence of p53 mutation in HPV-negative primary cervical carcinomas. The MDM2 oncogene, which forms an autoregulatory loop with the wild-type p53 protein, has been found amplified in a high percentage of human sarcomas, thus abolishing the antiproliferative function of p53. METHODS: Forty-three primary cervical carcinomas and 10 autopsy-derived distant metastases from one patient were examined for p53 mutation and MDM2 amplification. These tumors had been selected from 238 cervical cancers that had been HPV-typed by Southern blot hybridization and polymerase chain reaction as a representative sample for their HPV status and their clinicopathologic characteristics. Seventeen of the cases had a remarkably good or poor clinical outcome. Human papillomavirus DNA sequences had been detected in 30 of these 43 primary tumors and 13 were negative for HPV by both methods. p53 mutation in the highly conserved exons 5-8 was studied by single-strand conformation polymorphism analysis and direct sequencing. MDM2 amplification was analyzed by Southern blot hybridization. RESULTS: Only two missense point mutations and one nucleotide sequence polymorphism were detected: a TAC-->TGC transition in codon 234 in exon 7, resulting in a Tyr-->Lys substitution, a CGT-->TGT transition in codon 273 in exon 8, resulting in an Arg-->Cys substitution and a polymorphism (CGA-->CGG) in codon 213 in exon 6. Both tumors revealing the point mutations were HPV-negative carcinomas. Amplification of the MDM2 gene was observed in 1 of the 53 specimens tested. CONCLUSIONS: In contrast to data derived from cultured cervical carcinoma cell lines and primary sarcomas, these results indicate that p53 mutation and amplification of the MDM2 oncogene are rare even in HPV-negative primary cervical carcinomas. However, to the authors; knowledge, this is the first observation of MDM2 amplification in humans outside sarcomas and neuroepithelial tumors.     

Mutations of androgen receptor gene in Brazilian patients with male pseudohermaphroditism

We describe the identification of point mutations in the androgen receptor gene in five Brazilian patients with female assignment and behavior. The eight exons of the gene were amplified by the polymerase chain reaction (PCR) and analyzed for single-strand conformation polymorphism (SSCP) to detect the mutations. Direct sequencing of the mutant PCR products demonstrated single transitions in three of these cases: G-->A in case 1, within exon C, changing codon 615 from Arg to His; G-->A in case 2, within exon E, changing codon 752 from Arg to Gln, and C-->T in case 3, within exon B, but without amino acid change.     

A silent mutation, C924T (G308G), in the L1CAM gene results in X linked hydrocephalus (HSAS)

The L1 cell adhesion molecule (L1CAM) is a neuronal gene involved in the development of the nervous system. Mutations in L1CAM are known to cause several clinically overlapping X linked mental retardation conditions: X linked hydrocephalus (HSAS), MASA syndrome (mental retardation, aphasia, shuffling gait, adducted thumbs), spastic paraplegia type I (SPG1), and X linked agenesis of the corpus callosum (ACC). In an analysis of a family with HSAS, we identified a C-->T transition (C924T) in exon 8 that was initially thought to have no effect on the protein sequence as the alteration affected the third base of a codon (G308G). Extensive analysis of the other 27 exons showed no other alteration. A review of the sequence surrounding position 924 indicated that the C-->T transition created a potential 5' splice site consensus sequence, which would result in an in frame deletion of 69 bp from exon 8 and 23 amino acids of the L1CAM protein. RT-PCR of the RNA from an affected male fetus and subsequent sequence analysis confirmed the use of the new splice site. This is the first report of a silent nucleotide substitution in L1CAM giving rise to an alteration at the protein level. Furthermore, it shows that as mutation analysis plays an ever more important role in human genetics, the identification of a synonymous base change should not be routinely discounted as a neutral polymorphism.     

Electrocardiographic manifestations of dual atrioventricular node conduction during sinus rhythm

In search of critical genes associated with the mechanism of transforming growth factor-alpha (TGF alpha) action in human ovarian cancer, it was found that TGF alpha stimulates c-myc gene expression in human ovarian NIH:OVCAR-3. The role of c-myc in TGF alpha-stimulated growth of NIH:OVCAR-3 cells was examined by the use of the synthetic antisense-myc phosphorothioate oligonucleotide (OPT). Prior exposure of NIH:OVCAR-3 cells to an antisense-myc OPT inhibited TGF alpha-stimulated cell growth and DNA synthesis in a dose-dependent and sequence-specific manner over 4 days. c-Myc protein expression was down-regulated in the antisense-myc treated cells. These results demonstrate both the specific and durable effects of the antisense-myc OPT. Furthermore, the results suggest a role for c-myc in TGF alpha-stimulated cell proliferation.     

Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate

Recognition, internalization, and subcellular trafficking of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing N-acylated galactosamine (GalN) or monoclonal OV-TL16 antibodies (Ab) have been investigated in human hepatocarcinoma HepG2 and ovarian carcinoma OVCAR-3 cells, respectively. The intrinsic fluorescence of fluorescein or adriamycin (ADR) attached to HPMA copolymers permitted us to follow the subcellular fate of HPMA copolymer conjugates by confocal fluorescence microscopy and fluorescence spectroscopy. The pattern of fluorescence during incubation of HPMA copolymer-ADR-GalN conjugate containing lysosomally degradable tetrapeptide (GFLG) side-chains with HepG2 cells was consistent with conjugate recognition, internalization, localization in lysosomes, followed by the release of ADR from the polymer chains and ultimately diffusion via the cytoplasm into the cell nuclei. A similar pattern was observed in OVCAR-3 cells for Ab targeted HPMA copolymer conjugates. To test our hypothesis that HPMA-copolymer-bound anticancer drugs will be inaccessible to the energy-driven P-glycoprotein efflux pump in multidrug resistant (MDR) cells, we have compared the internalization of the HPMA copolymer-ADR conjugates by sensitive (A2780) and ADR-resistant (A2780/AD) ovarian carcinoma cell lines. Preliminary data on relative retention of ADR in MDR (A2780/AD) cells indicate a higher intracellular ADR concentration after incubation with HPMA copolymer-ADR conjugate when compared to incubation with free (unbound) ADR.     

Immunologic characterization of tumor markers in human ovarian cancer cell lines

OBJECTIVE: The purpose of this study was to evaluate the expression of a novel autologous ovarian tumor-associated antigen in eight human ovarian tumor cell lines compared with other ovarian tumor markers and products of oncogenes. METHODS: Autologous antibodies were eluted from human ovarian tumor-membrane fragments purified in our laboratory. These antibodies react with autologous ovarian tumor-associated antigens (AOTA) and have a high degree of specificity for human ovarian tumors. They do not bind to normal ovarian or nonovarian tissues, or to nonovarian neoplastic tissues. We evaluated eight human ovarian adenocarcinoma cell lines (2008, 2774, Caov-3, OVCAR-3, PA-1, SW 626, UCI 101, and UCI 107) by indirect immunofluorescence to determine the expression of AOTA relative to the ovarian cancer tumor marker CA 125 and the products of selected oncogenes (p 53, c-neu, and c-myc). RESULTS: The patterns and intensities of immunofluorescence correlated most closely between AOTA and c-neu. For example, AOTA and c-neu were detected in all eight cell lines and displayed very strong cytoplasmic fluorescence on cell lines 2774, UCI 101, and UCI 107. CA 125 was present in the cytoplasm of four of eight cell lines. A tumor suppressor gene product, p53, exhibited a nuclear staining pattern in six of eight cell lines. CONCLUSIONS: These data suggest that AOTA and the products of the c-neu oncogene may share certain epitopes. Current studies are underway to increase our understanding of the humoral response to ovarian cancer and the possible relationship to the expression of tumor oncogene products. Further characterization of AOTA will be necessary before early diagnostic tests can be developed.     

The usefulness of single-strand DNA conformation polymorphism analysis to detect mutations in protein C deficiency

The standard approach for the molecular genetic analysis of protein C deficiency, polymerase chain reaction (PCR) amplification followed by direct sequencing, although very accurate, is time-consuming. The aim of this study is to investigate the usefulness of a simplified, time-saving screening method for the detection of protein C mutations consisting of the combination of multiplex PCR amplifications using the same primers that were designed for sequencing, followed by single-strand DNA conformation polymorphism (SSCP) electrophoresis analysis performed with one set of conditions. The study was designed in two phases. First, we tested six known point mutations located in different exons of the protein C gene by SSCP. Second, we prospectively studied nine patients with protein C deficiency type I using SSCP as the first screening technique. All the exons were amplified with a common PCR protocol, either as single fragments or as multiplex combinations of several of them. In the retrospective study, three out of the six point mutations were visible as a band shift: 40 T-->G (exon 2), 1432 C-->T (exon 3) and 7253 C-->T (exon 8). In the prospective analysis SSCP detected three different mutations. These mutations were: 6128 T-->C (exon 7), 6216 C-->T (exon 7) and in two probands 8631 C-->T (exon 9). In the five remaining patients we identified only two different mutations by direct sequencing: 6246 G-->A (exon 7) in two patients and 8589 G-->A (exon 9) in four patients. In summary, the results from both studies show that only 60% of all mutations can be detected using this simplified method. It also suggests that a multiple set of conditions, smaller PCR fragments, or both, should be used in order to achieve a sensitivity comparable to sequencing.