Repression of BRCA1 through a feedback loop involving p53.

The BRCA1 and p53 tumor suppressors have been shown to interact and cooperate to activate transcription of p53-responsive genes. In this study, we show that BRCA1 is initially up-regulated, followed by a reduction to below basal levels in response to treatment with the DNA-damaging agents adriamycin and mitomycin C, and that the reduction of BRCA1 expression is dependent on the presence of wild-type p53. Elimination of p53 by expression of human papilloma virus E6 resulted in an inability to down-regulate BRCA1 in response to adriamycin. Ectopic expression of p53 resulted in a rapid decrease in BRCA1 protein and RNA levels and BRCA1 promoter-driven luciferase activity even in null p21 cells deficient in p53-dependent G(1) arrest. ATM(-)(/-) lymphoblastoid cells were deficient in their ability to reduce BRCA1 protein in response to DNA damage, whereas the wild-type counterparts reduced BRCA1 protein levels after exposure to adriamycin. These results, in conjunction with others, suggest a loop wherein BRCA1 initially participates in accumulation of p53 protein, whereas later p53 acts to reduce BRCA1 expression.

Detection of diverse variants of human immunodeficiency virus-1 groups M, N, and O and simian immunodeficiency viruses from chimpanzees by using generic pol and env primer pairs.

Human immunodeficiency virus type 1 (HIV-1) infection of humans is the result of independent cross-species transmissions of simian immunodeficiency viruses (SIVcpz) from naturally infected chimpanzees (Pan troglodytes troglodytes) to man. To develop a polymerase chain reaction-based assay capable of detecting members of all major phylogenetic SIVcpz and HIV-1 lineages (groups M, N, and O), primer pairs in conserved pol and env regions were designed. Both primer sets amplified 99%) in amplifying viral sequences from plasma taken from patients infected with HIV-1 group M (n=226) and O (n=17) viruses.

P53 tumor suppressor gene mutations in hepatocellular carcinoma patients in India.

BACKGROUND: Specific mutations of the p53 tumor suppressor gene in hepatocellular carcinoma (HCC) have been reported from several parts of the world, but to the authors' knowledge to date the status of this gene has not been studied in HCC patients in India, where HCC is one of the major cancers and the frequency of chronic hepatitis B virus (HBV) as well as hepatitis C virus (HCV) infection and exposure to dietary aflatoxin B(1) is very high. The most frequent mutation of the p53 gene in HCC is an AGG(Arg) to AGT(Ser) missense mutation at codon 249 of exon 7. METHODS: Liver biopsy specimens from 21 HCC patients and 10 healthy controls were obtained through surgery or by needle biopsy technique. Phenol-chloroform-extracted DNA specimens were employed for the detection of HBV infection and p53 gene mutations. Nucleotide mutations of exons 4-9 of the p53 gene were analyzed by polymerase chain reaction (PCR), single strand confirmation polymorphism, and direct sequencing. Third-generation sandwich enzyme-linked immunosorbent assay (ELISA) was used for the serologic detection of HBV and HCV infection. RESULTS: Analysis of exons 4-9 of the p53 gene revealed only 3 mutations (3 of 21 specimens, 14.28%; 95% confidence interval, -0.7-29.3), 2 mutations at codon 249 showing G-->T transversions, and 1 mutation (4.7%) at codon 250 with a C-->T transition. The base substitutions at the third base of codon 249 resulted in a missense mutation leading to a change in amino acid from arginine to serine whereas at codon 250 it caused a change from proline to serine. Dot blot hybridization and PCR for HBV DNA from HCCs revealed 58.8% (10 of 17 specimens) and 90. 47% (19 of 21 specimens), positivity, respectively. ELISA for hepatitis B virus surface antigen in serum showed a positivity of 71. 42% (15 of 21 specimens), but there was only 40% positivity (8 of 20 specimens) for hepatitis B virus envelope antigen whereas 6 of 17 patients (35.29%) showed the presence of antibodies against hepatitis B virus envelope protein. No patient was found to be positive for the HCV antibody. CONCLUSIONS: The very low frequency of p53 mutations and the extremely high frequency of HBV infection (> 90%) in HCC indicate that the mutations in the p53 gene frequently found in HCC reported from different endemic areas of the world may not play a direct role in the development of HCC in India. HBV infection and, possibly, exposure to the dietary aflatoxin B(1) appear to play major roles in the molecular pathogenesis of HCC in India.

Targeted cleavage of HIV-1 envelope gene by a DNA enzyme and inhibition of HIV-1 envelope-CD4 mediated cell fusion.

With the ultimate aim of developing an effective antiviral strategy against HIV-1, a mono-DNA enzyme possessing the 10-23 catalytic motif [Santoro and Joyce (1997) Proc. Natl. Acad. Sci. USA 94, 4264-4266] was synthesized against the HIV-1 envelope gene. We tested the in vitro cleavage efficiency of the 178 bp long truncated HIV-1 Env transcript by DNA enzyme 6339. Protein independent and Mg2+ dependent specific cleavage products were obtained. As soon as 5 min after mixing equimolar concentrations of DNA enzyme and substrate RNA, more than 50% cleavage was observed which increased steadily over a period of 4 h. Very little cleavage was obtained at 1 mM MgCl2 concentration which improved significantly when the concentration of MgCl2 was increased up to 20 mM. Specific inhibition of cell membrane fusion caused by the interaction of gp160 and CD4 in HeLa cells was observed when the above DNA enzyme was used. Thus, these chemically synthesized DNA enzymes could prove to be very useful for in vivo application.

Induction of chromosome aberrations and micronuclei in pulmonary alveolar macrophages of rats following inhalation of mosquito coil smoke.

The genotoxic potential of inhalation of mosquito coil (MC) smoke was evaluated by using metaphase chromosome aberration and micronucleus assays in pulmonary alveolar macrophages (PAMs) of rats following short-term as well as long-term whole body intermittent exposure. For short-term exposure, the animals were exposed for 15 min/h, 8 h/day to smoke collected for 1, 5 or 10 min, and they were killed 16 or 24 h after the final exposure. For long-term exposure, they were exposed for 15 min/h, 8 h/day, 7 days/week to smoke collected for 10 min and then they were killed 24 h after the final exposure. Each time before exposure, fresh smoke was collected by burning a mosquito coil. Pulmonary lavage was collected, and conventional flame-drying preparation was done for metaphase chromosome analysis and micronuclei (MN) were analyzed from smear preparations. Significantly higher frequencies of chromosome aberrations, including as well as excluding gaps, and micronucleated PAMs in smoke-exposed animals, compared to controls, indicated genotoxic capacity of MC smoke. The increases significantly correlated with the "concentration" of the gas. Mitotic indices also showed a significant and concentration-dependent increase. The frequencies of chromosome aberrations and MN following 7-day exposure were very similar to those for 1-day exposure. This was probably due to the transient nature of PAMs. A post-exposure gap of 24 h, compared to the 16-h gap, yielded a higher incidence of both mitoses and chromosome aberrations.

Genotoxicity of 'gudakhu', a tobacco preparation. II. In habitual users.

The genotoxic potential of 'gudakhu', a paste-like tobacco preparation that is used widely in Orissa, India, was evaluated using the micronucleus test in exfoliated cells of the buccal mucosa. Cells from 120 habitual users and from 102 non-users were examined. The incidence of micronuclei (MN) was increased in the mucosa cells of users, and the increase was significant in those who had used gudakhu for more than 5 yr. The increased incidence of MN was significantly correlated with the period of use of gudakhu, as well as with the frequency of daily use. There were no significant differences between the results for men and women.

Genotoxicity of 'gudakhu', a tobacco preparation. I. In mice in vivo.

'Gudakhu' is a paste-like tobacco preparation used widely in Orissa and neighbouring states of India. During use it is rubbed over the teeth and gum with a finger tip. Besides tobacco, it contains molasses, lime, red soil and water. The genotoxic potential of acetone extract of gudakhu was evaluated in mice in vivo using the chromosome aberration assay, micronucleus test and SCE analysis following single as well as long-term repeated treatment. The animals received an aqueous suspension of the extract via the oral route. Gudakhu extract induced significantly high frequencies, compared to controls, of chromosome aberrations, micronuclei (MN) and SCEs. Single treatment with different doses clearly revealed a distinct dose-dependent increase of the effects in all the assays. Analysis of MN in regenerated hepatocytes also indicated a significant positive correlation between time-course of chronic treatment and frequencies of micronucleated cells. But incidences of chromosome aberrations, MN and SCEs in bone marrow cells following repeated treatment for different periods did not differ greatly from each other; and these repeated treatment data, particularly in the MNT in bone marrow cells and the SCE assay, also did not differ markedly from the respective single treatment data for the same dose. This was probably due to the proliferative nature of the bone marrow cells.

Isolation of cDNA coding for an ubiquitous membrane protein deficient in high Na+, low K+ stomatocytic erythrocytes.

Human red blood cells (RBCs) that are deficient in an integral membrane-associated protein ("stomatin") of apparent molecular mass 31 Kd show a catastrophic increase in passive membrane permeability to the univalent cations Na+ and K+ and are stomatocytic in shape. We have purified this protein from normal RBC membranes and isolated a cDNA clone coding for it. The deduced protein sequence is unrelated to that of any known ion-transport-related protein. Selective solubilization studies using detergents show that while the protein is strongly associated with the phospholipid bilayer, it also binds to the cytoskeleton. The predicted polypeptide has a single trans-membranous hydrophobic segment near the N-terminus, which would locate it in the membrane; the large C-terminal domain is hydrophilic and cytoplasmic in orientation and is presumed to be responsible for the attachment to the cytoskeleton. By inference, the protein has the function of closing a latent ion channel. The messenger RNA encoding this protein is ubiquitously distributed in different human cell types and tissues and is thus presumably a widely distributed regulator of transmembrane cation fluxes. As a membrane-bound inhibitor protein of Na+ and K+ transport, it is unique among the known components of membrane-transport proteins.

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. III. Sister chromatid exchange analysis in bone marrow cells.

The potential genotoxicity of an antileprosy drug, clofazimine, was evaluated in mice in an in vivo model by sister chromatid exchange (SCE) analysis. Three different dose levels (4, 20 and 40 mg/kg) were tested, and the animals were treated once daily for 15 days. Sister chromatid differential staining was done by BrdU-tablet implantation and FPG technique. All the doses tested here elevated the SCE frequencies significantly and the increases showed a significant positive correlation with the doses. The results confirm our earlier findings based on metaphase analysis and micronucleus test in the same species.