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 B1 is very high. The most frequent mutation of the p53 gene in HCC is an AGGArg to AGTSer 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 B1 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

AbstractWith 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

Phosphorylation of p21 in G(2)/M Promotes Cyclin B-Cdc2 Kinase Activity

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G(2)/M phase. G(2)/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G(2)/M progression in p21(−/−) cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G(2)/M transition

Cell cycle checkpoint control mechanisms that can be disrupted in cancer

Maintenance of genomic integrity is essential to avoid cellular transformation, neoplasia, or cell death. DNA synthesis, mitosis, and cytokinesis are important cellular processes required for cell division and the maintenance of cellular homeostasis; they are governed by many extra- and intra-cellular stimuli. Progression of normal cell division depends on cyclin interaction with cyclin-dependent kinases (Cdk) and the degradation of cyclins before chromosomal segregation through ubiquitination. Multiple checkpoints exist and are conserved in the cell cycle in higher eukaryotes to ensure that if one fails, others will take care of genomic integrity and cell survival. Many genes act as either positive or negative regulators of checkpoint function through different kinase cascades, delaying cell cycle progression to repair the DNA lesions and breaks, and assuring equal segregation of chromosomes to daughter cells. Understanding the checkpoint pathways and genes involved in the cellular response to DNA damage and cell division events in normal and cancer cells, provides information about cancer predisposition, and suggests design of small molecules and other strategies for cancer therapy. Key Words: ATM-ATR; ATM/ATR; Aurora kinases; BRCAl; Cdc6; Cdc25; Cdc27-Cdc20/CdhI; Cell cycle; CENP-E; centrosome; checkpoint; Chkl/Chk2; cyc1in-Cdk; cyclindependent kinase inhibitors (CKI); hATRIP; Mad/Bub; MCM; MgcRacGAP; microtubule-associated proteins (MAPs); mitotic exit network (MEN); Mpsl; NIMA kinases; ORC; p53; PCNA; PBK-Akt; Plk; Rad50-Nbsl-Mrell; Ran-GTP; Ras; RB-E2F; SMC; Teml

Predominance of HIV type 1 subtype G among commercial sex workers from Kinshasa, Democratic Republic of Congo

We have investigated the genetic diversity and potential mosaic genomes of HIV-1 during the early part of the HIV-1 epidemic among commercial sex workers (CSWs) in Kinshasa, Democratic Republic of Congo (formerly Zaire). Serologic analysis revealed that 27 (28.7%) of the 94 specimens were seropositive by both peptide and whole-virus lysate EIAs and that 24 were positive by molecular screening assays, using generic primers that can detect all known groups of HIV-1. Phylogenetic analyses of the gag(p24), C2V3, and gp41 regions of these 24 specimens showed that all were group M; none of them had any evidence of group O, N, or SIVcpz-like sequences. On the basis of env sequence analysis, the 24 group M specimens were classified as subtypes G (37.5%), A (21%), F1 (12.5%), CRF01_AE (8%), D (4%), and H (4%); 3 (12.5%) were unclassifiable (U). Similar analysis of the gag(p24) region revealed that the majority of infections were subtype A; however, one-third of the specimens were subtype G. Parallel analysis of gag(p24) and env regions revealed discordant subtypes in many specimens that may reflect possible dual and/or recombinant viruses. These data suggest a predominance of subtype G (both pure G and recombinant CRF02_AG) during the early part of the epidemic in Kinshasa. Infections with group N or SIVcpz-like viruses were not present among these CSWs in Kinshasa