Screening cloned PCR fragments by restriction endonuclease finger-printing to obtain wild-type sequences.
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Bacteria↗
Biomedical subjects
C C Harris
Publications and source records attributed to C C Harris.
p21waf1/cip1 and transforming growth factor beta 1 protein expression correlate with survival in non-small cell lung cancer.
p21waf1/cip1 encodes a cyclin-dependent kinase inhibitor that is transcriptionally activated by the p53 tumor suppressor gene, transforming growth factor beta 1 (TGF-beta 1), AP2, and other pathways. Because p21waf1/cip1, p53, and TGF-beta 1 all regulate apoptosis and the cell cycle, we tested the hypothesis that their relative protein levels would correlate with biological features including the survival of non-small cell lung cancer (NSCLC) patients. We conducted an immunohistochemical analysis of p21waf1/cip1 and TGF-beta 1 and identified four patient groups with distinct survival outcomes. Concordant p21waf1/cip1 and TGF-beta 1 expression (i.e., either high p21waf1/cip1 and high TGF-beta 1 expression or low p21waf1/cip1 and low TGF-beta 1 expression) predicted 70% disease-free survival at 2000 days of follow-up. Discordant p21waf1/cip1 and TGF-beta 1 expression (i.e., either high p21waf1/cip1 and low TGF-beta 1 expression or low p21waf1/cip1 and high TGF-beta 1 expression) predicted 35% disease-free survival (P = 0.0003; log-rank test). These survival relationships were not attributable to differences in grade, stage, or p53 status. Although current models do not fully explain these complex interactions, most of these data fit a paradigm whereby TGF-beta 1 regulation determines NSCLC survival. In addition to the survival correlation, we found that high p21waf1/cip1 protein expression correlated with high tumor grade (P = 0.014). There is little evidence that p21waf1/cip1 protein levels accurately predict p53 mutation status in NSCLC; specifically, 20 of 48 (42%) tumors with p53 mutations contained high levels of p21waf1/cip1 protein. These findings indicate that p21waf1/cip1 immunohistochemical analysis may provide useful information concerning the biological properties of NSCLC.
Carcinoma, Non-Small-Cell Lung↗
Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis.
We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.
Adult↗
Interaction of p53 with the human Rad51 protein.
p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis. p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations. We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery. We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA. We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair. The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H. This binding is not mediated by a DNA or RNA intermediate. Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315. Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51. In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human. In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins. These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction.
Binding Sites↗
Genomic instability and telomerase activity in human bronchial epithelial cells during immortalization by human papillomavirus-16 E6 and E7 genes.
Human papilloma virus types 16 and 18 contribute to the development of cervical carcinomas in which the E6 and E7 genes are frequently retained and expressed in the tumors. Our study explored the ability of the E6 and/or E7 genes to immortalize normal human bronchial epithelial (NHBE) cells and to reactivate telomerase expression in these cells. We have introduced the human papillomavirus type 16 E6 or E7 genes alone or in combination (E6/E7) into NHBE cells using the retroviral construct pLXSN. Cells expressing either the E6 or the E7 oncoproteins alone displayed an increased colony-forming efficiency and a slightly extended in vitro life span before entering a crisis, from which immortalized cell lines were not obtained. Telomerase activity was not detected in cells expressing either E6 or E7 individually. Cells expressing the E6/E7 oncoproteins in combination had a substantially increased life span before entering crisis. A subpopulation of these cells escaped from crisis and achieved 130 population doublings, suggesting immortalization. Telomerase activity was detected in these postcrisis cells, but was not detected prior to crisis. In addition, karyotypic analysis showed evidence of genomic instability in mass cultures as well as clones expressing E6, E7, or E6/E7. Abnormalities included numerous monosomies and trisomies, chromatid gaps and breaks, double minutes, and aberrant chromosomes. These results demonstrate that expression of E6 and/or E7 is sufficient to induce genomic instability and an extended life span to NHBE cells, but the presence of both E6 and E7, along with at least one additional genetic or epigenetic event achieved during crisis, was required for reactivation of telomerase and the immortalization in this human cell type.
Bronchi↗
FHIT mutations in human primary gastric cancer.
Allelic deletion of multiple regions on the short arm of chromosome 3 (3p) implies the presence of multiple important tumor suppressor genes in human carcinogenesis. The FHIT gene, identified recently in chromosome 3p14.2, shows frequent allelic deletion and aberrant transcripts in gastrointestinal tumors. After determining the intron sequences flanking each of the coding exons of the FHIT gene and designing intron primers to facilitate mutation analysis of genomic DNA samples, we analyzed the complete coding sequences in matched cancer and normal tissues from 40 cases with primary gastric cancer using intron primers, PCR-single-strand conformation polymorphism analysis, and direct sequencing. A somatic missense mutation in exon 6, codon 61, ACG (threonine) --> ATG (methionine) was found in a signet ring cell adenocarcinoma. We also evaluated allelic deletion in these tumors by PCR-based microsatellite analysis; allelic deletion occurred in 42.1% (16 of 38) of evaluable cases. This is the first report of a somatic missense mutation of the FHIT gene in a primary tumor. Presence of a point mutation and frequent allelic deletions are consistent with the hypothesis that FHIT gene alterations are involved in the development of primary gastric cancers.
Acid Anhydride Hydrolases↗
Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers.
Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.
Base Sequence↗
Conformation-dependent phosphorylation of p53.
Phosphorylation of the p53 tumor suppressor protein is known to modulate its functions. Using bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53), we have determined human p53 phosphorylation by purified forms of jun-N-kinase (JNK), protein kinase A (PKA), and beta subunit of casein kinase II (CKIIbeta) as well as by kinases present in whole cell extracts (WCEs). We demonstrate that PKA is potent p53 kinase, albeit, in a conformation- and concentration-dependent manner, as concluded by comparing full-length with truncated forms of p53. We further demonstrate JNK interaction with GST-p53 and the ability of JNK to phosphorylate truncated forms of GST-p53 or full-length (His)p53. Dependence of phosphorylation on conformation of p53 is further supported by the finding that the wild-type form of p53 (p53wt) undergoes better phosphorylation by CKIIbeta and by WCE kinases than mutant forms of p53 at amino acid 249 (p53(249)) or 273 (p53(273)). Moreover, shifting the kinase reaction's temperature from 37 degrees C to 18 degrees C reduces the phosphorylation of mutant p53 to a greater extent than of p53wt. Comparing truncated forms of p53 revealed that the ability of CKIIbeta, PKA, or WCE kinases to phosphorylate p53 requires amino acids 97-155 within the DNA-binding domain region. Among three 20-aa peptides spanning this region we have identified residues 97-117 that increase p53 phosphorylation by CKIIbeta while inhibiting p53 phosphorylation by PKA or WCE kinases. The importance of this region is further supported by computer modeling studies, which demonstrated that mutant p53(249) exhibits significant changes to the conformation of p53 within amino acids 97-117. In summary, phosphorylation-related analysis of different p53 forms in vitro indicates that conformation of p53 is a key determinant in its availability as a substrate for different kinases, as for the phosphorylation pattern generated by the same kinase.
Adenosine Triphosphate↗
Integrity of p53 in hepatitis B x antigen-positive and -negative hepatocellular carcinomas.
Inactivation of the tumor suppressor p53 seems to be important to the pathogenesis of hepatocellular carcinoma (HCC) associated with chronic hepatitis B virus infection. Although this inactivation may be due to mutations in the p53 gene, recent evidence suggests that the hepatitis B virus-encoded X antigen (HBxAg) binds to and inactivates wild-type p53. Hence, experiments were designed to test the hypothesis that there is a low frequency of p53 mutations in HBxAg-positive HCC. HBxAg and p53 were assayed by immunohistochemistry (IHC) in HCC and nontumor liver from 16 Chinese patients, half of whom were hepatitis B surface antigen carriers. HBxAg was detectable in tumor and/or nontumor cells from all patients by IHC; six of these samples also had detectable p53. To determine whether p53 detection by IHC, and hence stabilization, is associated with mutation, sequencing of p53 exons 5-8 was performed with each patient sample. Wild-type sequences were found in 13 of 16 HBxAg-positive cases (81%). Hence, HBxAg is a common marker of HCC that correlates with the persistence of wild-type p53 among both carriers and noncarriers. The low frequency of p53 mutations in HCC in these patients implies that p53 inactivation may occur predominantly by complex formation with HBxAg.
Carcinoma, Hepatocellular↗
Database of p53 gene somatic mutations in human tumors and cell lines: updated compilation and future prospects.
In recent years, there has been an exponential increase in the number of p53 mutations identified in human cancers. The p53 mutation database consists of a list of point mutations in thep53 gene of human tumors and cell lines, compiled from the published literature and made available through electronic media. The database is now maintained at the International Agency for Research on Cancer (IARC) and is updated twice a year. The current version contains records on 5091 published mutations and is expected to surpass the 6000 mark in the January 1997 release. The database is available in various formats through the European Bioinformatics Institute (EBI) ftp server at: ftp://ftp.ebi.ac.uk/pub/databases/p53/ or by request from IARC (p53database@iarc.fr) and will be searchable through the SRS system in the near future. This report provides a description of the criteria for inclusion of data and of the current formats, a summary of the relevance ofp53 mutation analysis to clinical and biological questions, and a brief discussion of the prospects for future developments.
Databases, Factual↗
p53 tumor-suppressor gene: clues to molecular carcinogenesis.
The tumor-suppressor gene product p53 is clearly a component in several biochemical pathways, including transcription, DNA repair, genomic stability, cell-cycle control and apoptosis, that are central to human carcinogenesis. The p53 is functionally inactivated by mutational, viral, and cellular mechanisms in the majority of human cancers. Analysis of the spectrum of p53 mutations provides clues to the etiology and molecular pathogenesis of cancer. Recent insight into the p53-mediated biochemical pathways of cell-cycle arrest and apoptosis has provided further understanding of the mechanisms related to p53-mediated tumor suppression. This insight in turn may provide the potential molecular targets for the development of rational multimodality cancer therapy, including chemo-, immuno-, and gene-therapeutic strategies. The convergence of previously parallel lines of basic, clinical, and epidemiologic investigation may provide an opportunity to transfer research findings rapidly from the laboratory to the clinic.
Animals↗
Radon and lung carcinogenesis: mutability of p53 codons 249 and 250 to 238Pu alpha-particles in human bronchial epithelial cells.
Radon-222, a decay product of uranium-238 and a source of high linear energy transfer (LET) alpha-particles, has been implicated in the increased risk of lung cancer in uranium miners as well as non-miners. p53 mutation spectrum studies of radon-associated lung cancer have failed to show any specific mutational hot spot with the exception of a single study in which 31% of squamous cell and large cell lung cancers from uranium miners showed a p53 codon 249 AGGarg --> ATGmet mutation. Although the results of laboratory studies indicate that double-strand breaks and deletions are the principal genetic alterations caused by alpha-particles, uncertainty still prevails in the description of DNA damage in radon-associated human lung cancer. In the present study, we have evaluated the mutability of p53 codons 249 and 250 to alpha-particles in normal human bronchial epithelial (NHBE) cells using a highly sensitive genotypic mutation assay. Exposure of NHBE cells to a total dose of 4 Gy (equivalent to approximately 1460 working level months in uranium mining) of high LET alpha-radiation induced codon 249 AGG --> AAG transitions and codon 250 CCC --> ACC transversions with absolute mutation frequencies of 3.6 x 10(-7) and 3.8 x 10(-7) respectively. This mutation spectrum is consistent with our previous report of radon-associated human lung cancer.
Adolescent↗
Infrequent mutations of the transforming growth factor beta-type II receptor gene at chromosome 3p22 in human lung cancers with chromosome 3p deletions.
Mutations in the transforming growth factor beta-type II receptor (TGFbeta RII) gene have been detected in several types of human cancers that represent the phenotype of genomic instability. The TGFbeta RII gene has been mapped to chromosome 3p, on which loss of heterozygosity (LOH) was frequently detected in both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). To investigate whether the TGFbeta RII gene on 3p22 is inactivated in lung cancers, we examined 35 sporadic lung cancers (15 SCLC and 20 NSCLC) with LOH on 3p for mutations of the TGFbeta RII gene. We previously produced eight intron based primer pairs for mutational analysis of the entire coding region of the TGFbeta RII gene. Using these primers, we screened for mutations of the TGFbeta RII gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. A mutation was detected in a case of SCLC: one base insertion in the polyadenine tract of exon 3. This tumor showed the replication error (RER) phenotype. There were no mutations in exons 1, 2, 4, 5, 6 and 7. These results indicate that the polyadenine tract is a mutational hot spot in the TGFbeta RII gene in RER positive tumors, and that TGFbeta RII mutations occur rarely in lung cancers with LOH on chromosome 3p.
Carcinoma, Non-Small-Cell Lung↗
Serum, plasma and paraffin-embedded tissues as sources of DNA for studying cancer susceptibility genes.
The ability to isolate DNA from archived human serum, plasma and paraffin-embedded human tissues enhances opportunities to study breast, lung and other cancer risk factors. We report herein a simple and fast protocol for the extraction of genomic DNA from these sources. Using a phenol-based extraction method, the recovery for DNA is quantitative and reproducible. DNA yields in serum (250 microl) were between 162 and 1060 ng (n = 18 subjects), in plasma (250 microl) were between 165 and 375 ng (n = 5 subjects) and in embedded tissues (5-microm thick sections for ethanol fixed, and between 5- and 20-microm sections for formaldehyde fixation) were between 1 microg and 11.7 microg (n = 32 subjects). The extraction method was combined with newly designed PCR-based assays for cancer susceptibility marker genes such as CYP1A1 (exon 7), CYP2E1 (Dra1, Rsa1), GSTM1 and NAT2 [NAT2*5A (C481T), NAT2*6A (G590A), NAT2*7A (G857A)]. Genotyping results from the serum and paraffin-embedded tissues compared favorably to results from archived freshly frozen tissues, where concordance was 98% for serum, 100% for ethanol-fixed embedded tissues, and 97% for formaldehyde-fixed and paraffin-embedded tissues. This facile method will allow for the use of archived tissue samples of prospective cohort and other studies where intact DNA was not previously available.
Arylamine N-Acetyltransferase↗
Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines.
Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.
Adult↗
Absence of mutations in the transforming growth factor-beta type II receptor in sporadic lung cancers with microsatellite instability and rare H-ras1 alleles.
The transforming growth factor-beta type II receptor (RII) is commonly mutated in colon and gastric cancers with microsatellite instability (MI). We utilized our series of lung cancers with MI and rare alleles of the H-ras1 gene to determine the association between MI and RII mutations and searched the entire RII coding region in 33 lung cancers with MI by polymerase chain reaction-single-strand conformation polymorphism analysis. We found no mutations, and these data support other recent evidence that RII mutations rarely occur except in colon and gastric tumors with MI.
Alleles↗
Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression.
The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage. DNA damage can lead to p53-mediated growth arrest and apoptosis. High concentrations of nitric oxide (NO) and NO metabolites such as peroxynitrite and NO2 cause DNA damage and have been shown to be mutagenic. Furthermore, NO induces p53 accumulation and, as part of a feedback loop, p53 mediates transcriptional transrepression of inducible nitric oxide synthase. Recent studies have shown increased expression and activity of nitric oxide synthase isoforms in human cancer. NO has both genotoxic and angiogenic properties, so that increased NO production may select mutant p53 cells and contribute to human carcinogenesis and tumor progression.
Animals↗
Differential effects of p53 mutants on the growth of human bronchial epithelial cells.
We investigated the effects of five different p53 mutants on the growth of primary cultures of normal human bronchial epithelial (NHBE) cells. The five defective viral pZIP-Neo constructs contained the following mutations at mutational hot-spots found in human cancers: codons 143ala, 175his, 248trp, 249ser, and 273his. NHBE cells were infected with the p53 mutants, wild-type p53, or the pZIP-Neo vector control. The 143ala, 248trp, and 273his mutants, as well as wild-type p53, decreased the colony-forming efficiency and inhibited the growth of NHBE cells. The 175his mutant did not significantly change the growth rates. In NHBE cells from three donors, the 249ser mutant conferred a substantial growth advantage to the NHBE cells in a colony-forming-efficiency assay. In NHBE cells isolated from one donor, the 249ser mutant also produced a significant life span extension. These cells grew rapidly through 80 population doublings and entered an apparent "crisis" in passage 14. Karyotypic analyses of one culture at multiple passages revealed aneuploid populations with alterations of chromosomes 5, 11, and 13; quantitative DNA analysis detected aneuploidy in late passages from that culture and two other primary cultures. These data demonstrated that the codon 249ser mutation could provide a growth advantage to bronchial epithelial cells and suggest that this mutant protein can induce genomic instability.
Bronchi↗