Gene Therapy with p14/tBID Induces Selective and Synergistic Apoptosis in Mutant Ras and Mutant p53 Cancer Cells In Vitro and In Vivo.

Any gene therapy for cancer will be predicated upon its selectivity against cancer cells and non-toxicity to normal cells. Therefore, safeguards are needed to prevent its activation in normal cells. We designed a minimal p14ARF promoter with upstream Ap1 and E2F enhancer elements and a downstream MDR1 inhibitory element, TATA box, and a transcription initiation site (hereafter p14ARFmin). The modified p14ARFmin promoter was linked to bicistronic P14 and truncated BID (tBID) genes, which led to synergistic apoptosis via the intrinsic and extrinsic pathways of apoptosis when expressed. The promoter was designed to be preferentially activated by mutant Ras and completely inhibited by wild-type p53 so that only cells with both mutant Ras and mutant p53 would activate the construct. In comparison to most p53 gene therapies, this construct has selective advantages: (1) p53-based gene therapies with a constitutive CMV promoter cannot differentiate between normal cells and cancer cells, and can be toxic to normal cells; (2) our construct does not induce p21WAF/CIPI in contrast to other p53-based gene therapies, which can induce cell cycle arrest leading to increased chemotherapy resistance; (3) the modified construct (p14ARFmin-p14-tBID) demonstrates bidirectional control of its promoter, which is completely repressed by wild-type p53 and activated only in cells with both RAS and P53 mutations; and (4) a novel combination of genes (p14 and tBID) can synergistically induce potent intrinsic and extrinsic apoptosis in cancer cells.

C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells In Vitro and In Vivo.

We previously demonstrated that a synthetic monomer peptide derived from the C-terminus of p53 (aa 361−382) induced preferential apoptosis in mutant p53 malignant cells, but not normal cells. The major problem with the peptide was its short half-life (half-life < 10 min.) due to a random coil topology found in 3D proton NMR spectroscopy studies. To induce secondary/tertiary structures to produce more stability, we developed a peptide modelled after the tetrameric structure of p53 essential for activation of target genes. Starting with the above monomer peptide (aa 361−382), we added the nuclear localization sequence of p53 (aa 353−360) and the end of the C-terminal sequence (aa 383−393), resulting in a monomer spanning aa 353−393. Four monomers were linked by glycine to maximize flexibility and in a palindromic order that mimics p53 tetramer formation with four orthogonal alpha helices, which is required for p53 transactivation of target genes. This is now known as the 4 repeat-palindromic-p53 peptide or (4R-Pal-p53p). We explored two methods for testing the activity of the palindromic tetrapeptide: (1) exogenous peptide with a truncated antennapedia carrier (Ant) and (2) a doxycycline (Dox) inducer for endogenous expression. The exogenous peptide, 4R-Pal-p53p-Ant, contained a His tag at the N-terminal and a truncated 17aa Ant at the C-terminal. Exposure of human breast cancer MB-468 cells and human skin squamous cell cancer cells (both with mutant p53, 273 Arg->His) with purified peptide at 7 µM and 15 µM produced 52% and 75%, cell death, respectively. Comparatively, the monomeric p53 C-terminal peptide-Ant (aa 361−382, termed p53p-Ant), at 15 µM and 30 µM induced 15% and 24% cell death, respectively. Compared to the p53p-Ant, the exogenous 4R-pal-p53p-Ant was over five-fold more potent for inducing apoptosis at an equimolar concentration (15 µM). Endogenous 4R-Pal-p53p expression (without Ant), induced by Dox, resulted in 43% cell death in an engineered MB468 breast cancer stable cell line, while endogenous p53 C-terminal monomeric peptide expression produced no cell death due to rapid peptide degradation. The mechanism of apoptosis from 4R-Pal-p53p involved the extrinsic and intrinsic pathways (FAS, caspase-8, Bax, PUMA) for apoptosis, as well as increasing reactive oxygen species (ROS). All three death pathways were induced from transcriptional/translational activation of pro-apoptotic genes. Additionally, mRNA of p53 target genes (Bax and Fas) increased 14-fold and 18-fold, respectively, implying that the 4R-Pal-p53p restored full apoptotic potential to mutant p53. Monomeric p53p only increased Fas expression without a transcriptional or translational increase in Fas, and other genes and human marrow stem cell studies revealed no toxicity to normal stem cells for granulocytes, erythrocytes, monocytes, and macrophages (CFU-GEMM). Additionally, the peptide specifically targeted pre-malignant and malignant cells with mutant p53 and was not toxic to normal cells with basal levels of WT p53.

Effects of DNA repair gene polymorphisms on DNA damage in human lymphocytes induced by a vinyl chloride metabolite in vitro.

BACKGROUND: Epidemiologic studies suggest that variability in DNA damage from vinyl chloride monomer (VCM) may be partially mediated by genetic polymorphisms in DNA repair. This study aimed to corroborate these observations with controlled experiments in vitro using cell lines from individuals with differing DNA repair genotypes to determine damage following VCM metabolite exposure. METHODS: Matched pairs of lymphoblast cell lines (homozygous wild-type versus homozygous variant for either XRCC1 399 or XPD 751 polymorphism) were exposed to chloroacetaldehyde and analyzed by the cytokinesis-block micronucleus assay. RESULTS: All cell lines demonstrated a dose-response of increasing micronuclei with increasing exposure, but for both XRCC1 and XPD, the polymorphic cells peaked at higher micronucleus frequencies and declined at a slower rate to baseline than the wild-type cells. CONCLUSION: This supports the findings that XRCC1 and XPD polymorphisms may result in deficient DNA repair of VCM-induced genetic damage.

Polymorphisms in BER and NER pathway genes: effects on micronucleus frequencies among vinyl chloride-exposed workers in Northern China.

In this study, a group of 317 workers occupationally exposed to vinyl chloride monomer and 166 normal, unexposed referents in Shandong province (Northern China) were examined for chromosomal damage in peripheral lymphocytes using the cytokinesis-blocked micronucleus (CB-MN) assay. The exposure group (3.47±2.65)‰ showed higher micronucleus frequency than the unexposed workers (2.51±1.96)‰ (P<0.01). We explored the relationship between genetic polymorphisms of XRCC1 (-77C/T, Arg194Trp, Arg280His, Arg399Gln), APE1 Asp148Glu, XPA Ala23Gly, XPC.PAT, XPC Ala499Val, XPC Lys939Gln, XPF 5'-UTR T2063A, XPG Exon15 G-C, ERCC13'-UTR C8092A and susceptibility of chromosomal damage in all the subjects. It was found that XRCC1 -77, XRCC1 280, APE1148, XPC.PAT, XPG Exon15 G-C, and ERCC13'-UTR C8092A polymorphisms showed no significant associations with micronucleus frequency in unexposed workers. However, among the exposed workers individuals with XRCC1 (-77C/T, Arg194Trp, Arg280His, Arg399Gln) polymorphisms had a significantly higher micronucleus frequency as seen in mean frequency ratios (FR) compared with their homozygous wild-type genotypes (FR=1.21, 95% CI: 1.05-1.39; P<0.01); (FR=1.14, 95% CI: 1.00-1.38; P<0.05) and (FR=1.26, 95% CI: 1.11-1.44; P<0.01); (FR=1.23, 95% CI: 1.08-1.46; P<0.01). Four SNP sites in the nucleotide excision repair (NER) pathway were associated with susceptibility for MN frequency in either unexposed or exposed workers. Further, we observed the gene-MN association changed with exposure for XRCC1 (-77C/T, Arg194Trp, Arg280His, Arg399Gln), XPA Ala23Gly, XPC Ala499Val, XPC Lys939Gln, XPF 5'-UTR T2063A. Moreover, Individuals carrying the XPC (PAT)-(499)-(939) diplotype, PAT-CG/PAT-TG, had a higher MN frequency, compared with individuals carrying the wild-type PAT-CA/PAT-CA.

Correlation of chromosome damage and promoter methylation status of the DNA repair genes MGMT and hMLH1 in Chinese vinyl chloride monomer (VCM)-exposed workers.

OBJECTIVE: To explore the association of the methylation status of MGMT and hMLH1 with chromosome damage induced by vinyl chloride monomer (VCM). MATERIALS AND METHODS: Methylation of MGMT and hMLH1 was measured in 101 VCM-exposed workers by methylation-specific PCR. Chromosome damage in peripheral blood lymphocytes was measured by the cytokinesis-block micronucleus assay. The subjects were divided into chromosome damaged and non-damaged groups based on the normal reference value of micronuclei frequencies determined for two control groups. RESULTS: MGMT promoter methylation was detectable in 5 out of 49 chromosome damaged subjects, but not in the chromosome non-damaged subjects; there was a significant difference in MGMT methylation between the two groups (p < 0.05). CONCLUSIONS: We detected aberrant promoter methylation of MGMT in a small number of chromosome damaged VCM-exposed workers, but not in the chromosome non-damaged subjects. This preliminary observation warrants further investigation in a larger study.

Estimation of benchmark dose for micronucleus occurrence in Chinese vinyl chloride-exposed workers.

In this study, we estimated the possibility of using benchmark dose (BMD) to assess the dose-response relationship between vinyl chloride monomer (VCM) exposure and chromosome damage. A group of 317 workers occupationally exposed to vinyl chloride monomer and 166 normal, unexposed control in Shandong Province northern China were examined for chromosomal damage in peripheral blood lymphocytes (PBL) using the cytokinesis-blocked micronucleus (CB-MN) assay of DNA damage. The exposed group (3.47 ± 2.65)‰ showed higher micronucleus frequency than the control (1.60 ± 1.30)‰ (P < 0.01). Occupational exposure level based on micronucleus occurrence in all individuals was analyzed with benchmark dose (BMD) methods. The benchmark dose lower limit of a one-sided 95% confidence interval (BMDL) for 10% excess risk was also determined. Results showed a dose-response relationship between cumulative exposure and MN frequency, and a BMDL of 0.54 mg/m3 and 0.23 mg/m3 for males and females, respectively. Female workers were more susceptible to MN damage than male workers.

Polymorphisms in the p53 pathway genes and micronucleus occurrence in Chinese vinyl chloride-exposed workers.

OBJECTIVES: To investigate the association between polymorphisms in the p53 pathway genes and chromosomal damage in vinyl chloride (VC)-exposed workers. MATERIALS AND METHODS: Cytokinesis block micronucleus test was performed in 310 VC-exposed workers and 149 non-exposed workers to determine chromosomal damage. The polymerase chain reaction and restriction fragment length polymorphism technique were used to detect six SNPs in the p53 pathway genes involved in the cell cycle. RESULTS: There was a highly significant dose-response relationship between VC exposure and chromosomal damage. Individuals carrying the variant genotypes were at higher risk for chromosomal damage compared with their wild type genotype: p53rs1042522, MDM2 Del1518rs3730485, MDM2rs2279744 and GADD45Ars532446. On the other hand, individuals possessing the variant genotype of CDKN2A rs3088440 had significantly decreased risk compared with the corresponding wild-type. CONCLUSIONS: Genetic polymorphisms in P53 pathway genes may have an impact on VC-induced chromosomal damage.

Estimation of a safe level for occupational exposure to vinyl chloride using a benchmark dose method in central China.

OBJECTIVES: The aim of this study was to estimate a benchmark dose (BMD) for chromosome damage induced by vinyl chloride monomer (VCM) in VCM-exposed workers in central China and validate the published results in Shanghai. METHODS: VCM-exposed workers who had been exposed to VCM for at least one year (n=463) and matched subjects not exposed to VCM or other toxins (n=273) were asked to participate in this study. Micronucleus (MN) frequency based on the cytokinesis-block micronucleus assay (CBMN) was used as a biomarker for chromosome damage induced by VCM exposure. RESULTS: The MN frequency in the VCM-exposed workers was significantly higher than that in the control group, and multivariate Poisson regression suggested that gender, smoking status and VCM exposure were the significant factors influencing the risk of increased MN frequency. When subjects were further stratified according to gender and smoking status, the results showed that female VCM-exposed workers were more susceptible than the males to the risk of increased MN frequency. The MN frequency of smokers was significantly higher than that of nonsmokers in the control group. Our study also suggested that there was a strong dose-response relationship between VCM CED and the increased risk of MN frequency in the total group, males and females. The BMDL(10) was found to be 630.6, 670.2 and 273.7 mg-year for all VCM-exposed workers, males and females, respectively. CONCLUSIONS: These results invite further scrutiny of the current VCM occupational exposure limits and warrant further study of the risk of VCM genotoxicity and carcinogenicity.

The differing perspectives of workers and occupational medicine physicians on the ethical, legal and social issues of genetic testing in the workplace.

Genetic testing in the workplace holds the promise of improving worker health but also raises ethical, legal, and social issues. In considering such testing, it is critical to understand the perspectives of workers, who are most directly affected by it, and occupational health professionals, who are often directly involved in its implementation. Therefore, a series of focus groups of unionized workers (n=25) and occupational medicine physicians (n=23) was conducted. The results demonstrated strikingly different perspectives of workers and physicians in several key areas, including the goals and appropriateness of genetic testing, and methods to minimize its risks. In general, workers were guided by a profound mistrust of the employer, physician, and government, while physicians were guided primarily by scientific and medical concerns, and, in many cases, by the business concerns distrusted by the workers.

Proteomic detection of cancer in asbestosis patients using SELDI-TOF discovered serum protein biomarkers.

OBJECTIVES: To identify biomarkers for cancer in asbestosis patients. METHODS: SELDI-TOF and CART were used to identify serum biomarker profiles in 35 asbestosis patients who subsequently developed cancer and 35 did not develop cancer. RESULTS: Three polypeptide peaks (5707.01, 6598.10, and 20,780.70 Da) could predict the development of cancer with 87% sensitivity and 70% specificity. The first two peaks were identified as KIF18A and KIF5A, respectively, and are part of the Kinesin Superfamily of proteins. CONCLUSIONS: We identified two Kinesin proteins that can be potentially used as blood biomarkers to identify asbestosis patients at risk of developing lung cancer.

Protein and amino acid intakes in a rural area of Bangladesh.

BACKGROUND: Few studies have described protein and amino acid intakes in rural Bangladesh, a country with considerable undernutrition. OBJECTIVE: The purpose of this population-based study was to assess and describe protein and amino acid intakes in Araihazar, Bangladesh. METHODS: The study participants were 11,170 adult men and women who participated in the Health Effects of Arsenic Longitudinal Study (HEALS), which had a 98% participation rate. Dietary exposures were assessed by a food-frequency questionnaire that had been designed and validated for the HEALS study population. RESULTS: The mean body mass index (BMI) was 19.7 among all participants, and 34.9% of women and 44.4% of men had a BMI below 18.5. The average caloric intake was 2142 and 2394 kcal/day among women and men, respectively, and the mean protein intake was 67.5 and 78.2 g/day. The largest sources of protein were from rice and fish. Greater protein intake was related to younger age and several socioeconomic measures, including more years of education, land and television ownership, and employment in business, farming, or as a laborer (for men) or as a homemaker (for women). CONCLUSIONS: This study found a high prevalence of underweight among study participants. Nonetheless, most participants had adequate protein intake according to Food and Agriculture Organization standards for body weight.

A prospective study of respiratory symptoms associated with chronic arsenic exposure in Bangladesh: findings from the Health Effects of Arsenic Longitudinal Study (HEALS).

BACKGROUND AND AIMS: A prospective cohort study was conducted to evaluate the effect of arsenic (As) exposure from drinking water on respiratory symptoms using data from the Health Effects of Arsenic Exposure Longitudinal Study (HEALS), a large prospective cohort study established in Ariahazar, Bangladesh in 2000-2002. A total of 7.31, 9.95 and 2.03% of the 11 746 participants completing 4 years of active follow-up reported having a chronic cough, breathing problem or blood in their sputum, respectively, as assessed by trained physicians. METHODS: Cox regression models were used to estimate HRs for respiratory symptoms during the follow-up period in relation to levels of chronic As exposure assessed at baseline, adjusting for age, gender, smoking, body mass index, education and arsenic-related skin lesion status. RESULTS: Significant positive associations were found between As exposure and respiratory symptoms. As compared with those with the lowest quintile of water As level (178 microg/l), respectively. Similarly, the corresponding HRs in relation to the second to fifth quintiles of urinary arsenic were 1.10 (95% CI 0.94 to 1.27), 1.11 (95% CI 0.95 to 1.29), 1.29 (95% CI 1.11 to 1.49) and 1.35 (95% CI 1.16 to 1.56), respectively. These associations did not differ appreciably by cigarette smoking status. CONCLUSIONS: This prospective cohort study found a dose-response relationship between As exposure and clinical symptoms of respiratory diseases in Bangladesh. In particular, these adverse respiratory effects of As were clearly evident in the low to moderate dose range, suggesting that a large proportion of the country's population may be at risk of developing serious lung diseases in the future.

Genetic polymorphisms of XRCC1, HOGG1 and MGMT and micronucleus occurrence in Chinese vinyl chloride-exposed workers.

In this study, a group of 313 workers occupationally exposed to vinyl chloride monomer (VCM) and 141 normal unexposed referents were examined for chromosomal damage using the cytokinesis-blocked micronucleus (CBMN) assay in peripheral lymphocytes. We explored the relationship between genetic polymorphisms of XRCC1 (Arg194Trp, Arg280His and Arg399Gln), MGMT(Leu84Phe) and hOGG1 (Ser326Cys) and susceptibility of chromosomal damage induced by VCM. Polymerase chain reaction-restriction fragment length polymorphism techniques were used to detect polymorphisms in XRCC1, hOGG1 and MGMT. It was found that the micronuclei (MN) frequency of exposed workers (4.86 +/- 2.80) per thousand was higher than that of the control group (1.22 +/- 1.24) per thousand (P < 0.01). Increased susceptibility to chromosomal damage as evidenced by higher MN frequency was found in workers with hOGG1 326 Ser/Cys genotype [frequency ratio (FR) = 1.21, 95% confidence interval (CI): 1.02-1.46; P < 0.05], XRCC1 194 Arg/Trp (FR = 1.12, 95% CI: 1.00-1.25; P < 0.05) and XRCC1 280 Arg/His and His/His genotypes (FR = 1.12, 95% CI 1.00-1.26, P < 0.05). Moreover, among susceptibility diplotypes, CGA/CAG carriers had more risk of MN frequency compared with individuals with wild-type CGG/CGG (FR = 1.67, 95% CI: 1.19-2.23; P < 0.05). MN frequency also increased significantly with age in the exposed group (FR = 1.13, 95% CI: 1.00-1.28; P < 0.05). Thus, CB-MN was a sensitive index of early damage among VCM-exposed workers. Genotype XRCC1 Arg194Trp, Arg280His, hOGG1 Ser326Cys, diplotype CGA/CAG and higher age may have an impact on the chromosome damage induced by VCM.

Genetic polymorphisms in metabolizing enzymes and susceptibility of chromosomal damage induced by vinyl chloride monomer in a Chinese worker population.

OBJECTIVE: To evaluate whether polymorphisms in metabolizing enzymes contributed to susceptibility of chromosomal damage induced by vinyl chloride monomer (VCM). METHODS: Cytokinesis block micronucleus test was performed on 185 VCM-exposed workers and 41 control subjects to detect chromosomal damage in peripheral lymphocytes. The polymerase chain reaction and restriction fragment length polymorphism technique was applied to detect polymorphisms of GSTT1, GSTM1, GSTP1G/A, CYP2E1G/C, and CYP2D6G/C. Poisson regression analysis was performed. RESULTS: Sex, age, VCM exposure, GSTP1, and CYP2E1 genotype can influence chromosomal damage. There was a 1.51-fold increased micronucleus frequency for GSTP1GG genotypes individuals compared with those GSTP1AA/GA genotype individuals (P < 0.05), the effect of polymorphism in CYP2E1 gene was more pronounced for allele C compared with allele G (P < 0.05). CONCLUSIONS: Polymorphisms of GSTP1G/A and CYP2E1G/C, which are potential susceptibility biomarkers of chromosomal damage in VCM-exposed worker.

Effect of the XRCC1 codon 399 polymorphism on the repair of vinyl chloride metabolite-induced DNA damage.

BACKGROUND: Recent epidemiologic evidence suggests that the common polymorphism at amino acid residue 399 of the x-ray cross complementing-1 (XRCC1) protein, a key component of the base excision repair (BER) pathway for DNA damage, plays a significant role in the genetic variability of individuals in terms of the mutagenic damage they experience following exposure to the carcinogen vinyl chloride (VC). The aim of this study was to provide support for the biological plausibility of these epidemiologic observations with experimental data derived from cell lines in culture from individuals who were either homozygous wild-type or homozygous variant for this XRCC1 polymorphism following exposure to chloroethylene oxide (CEO), the active metabolite of VC, with measurement of the induced etheno-DNA adducts before and after repair. MATERIALS AND METHODS: Immortalized lymphoblast cell lines from seven VC workers (four homozygous wild-type and three homozygous variant for the 399 XRCC1 polymorphism) were exposed to CEO, and etheno-adenosine (epsilonA) adduct levels were determined by enzyme-linked immunosorbent assay (ELISA) pre-exposure and at 0, 4, 8 and 24 h following exposure. RESULTS: The average epsilonA adduct levels were statistically significantly higher in the variant cells compared to the wild-type cells at 8 and 24 h following exposure (P Conclusion: These results are consistent with the epidemiologic findings of the types of VC-induced biomarkers observed in exposed individuals and the mutational spectra found in the resultant tumors as well as the key role that BER, especially XRCC1, plays in this carcinogenic pathway.

Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein.

AIM: The xeroderma pigmentosum D (XPD) protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER) and transcription-coupled repair (TCR). The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln). Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. MATERIALS AND METHODS: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. RESULTS: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. CONCLUSION: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.