HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

Bcl-2 overexpression in PhIP-induced colon tumors: cloning of the rat Bcl-2 promoter and characterization of a pathway involving beta-catenin, c-Myc and E2F1.

Beta-catenin/T-cell factor (Tcf) signaling is constitutively active in the majority of human colorectal cancers, and there are accompanying changes in Bcl-2 expression. Similarly, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP)-induced colon tumors in the rat have increased beta-catenin and elevated Bcl-2. To examine the possible direct transcriptional regulation of rat Bcl-2 by beta-catenin/Tcf, we cloned and characterized the corresponding promoter region and found 70.1% similarity with its human counterpart, BCL2. Bcl-2 promoter activity was increased in response to LiCl and exogenous beta-catenin, including oncogenic mutants of beta-catenin found in PhIP-induced colon tumors. Protein/DNA arrays identified E2F1, but not beta-catenin/Tcf, as interacting most strongly with the rat Bcl-2 promoter. Exogenous E2F1 increased the promoter activity of rat Bcl-2, except in mutants lacking the E2F1 sites. As expected, beta-catenin induced its downstream target c-Myc, as well as E2F1 and Bcl-2, and this was blocked by siRNA to c-Myc or E2F1. These findings suggest an indirect pathway for Bcl-2 over-expression in PhIP-induced colon tumors involving beta-catenin, c-Myc and E2F1.

Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.

Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.

Genotype and allele frequency of a 32-base pair deletion mutation in the CCR5 gene in various ethnic groups: absence of mutation among Asians and Pacific Islanders.

BACKGROUND: A 32-base pair (bp) deletion mutation in the beta-chemokine receptor CCR5 gene has been associated with resistance against human immunodeficiency virus type 1 (HIV-1) infection and disease. Large-scale studies conducted among Caucasians indicate that individuals who are homozygous for this deletion mutation (D32/D32) are protected against HIV-1 infection despite multiple high-risk exposures, whereas CCR5/ D32 heterozygotes have a slower progression to acquired immunodeficiency syndrome (AIDS). OBJECTIVE: To determine the genotype and allele frequencies of the CCR5 gene 32-bp deletion mutation among ethnically diverse non-Caucasian populations. METHODS: DNA, extracted from blood collected between 1980 and 1997 from 1912 individuals belonging to various ethnic groups, including 363 Caucasians, 303 Puerto Rican Hispanics, 150 Africans, 606 Asians, and 490 Pacific Islanders, were analyzed for the CCR5 gene 32-bp deletion mutation by a polymerase chain reaction (PCR)-based assay, using an oligonucleotide primer pair designed to discriminate CCR5 alleles without restriction endonuclease analysis. RESULTS: The comparative frequency of CCR5/D32 heterozygosity was 61 of 363 (16. 8%) in Caucasians, 17 of 303 (5.6%) in Puerto Rican Hispanics, 9 of 490 (1.8%) in Pacific Islanders, 0 of 606 (0%) in Asians, and 0 of 150 (0%) in Africans. CONCLUSIONS: The data confirm the high frequency of CCR5/D32 heterozygosity among Caucasians. Intermediate and low-level D32 allele frequencies among Puerto Rican Hispanics and Hawaiians could be attributed to recent European Caucasian gene flow. By contrast, the inability to detect the D32 allele among Asians and other Pacific Islander groups suggests that other mechanisms are responsible for resistance to HIV-1 infection in these populations.

High prevalence of GB virus C/hepatitis G virus infection among homosexual men infected with human immunodeficiency virus type 1: evidence for sexual transmission.

GB virus C/hepatitis G virus (GBV-C/HGV), a recently discovered orphan flavivirus, is distantly related to hepatitis C virus (HCV). Although both GBV-C/HGV and HCV can be transmitted by the parenteral route, their principal modes of transmission and associated risk behaviors may differ. Using reverse transcription-polymerase chain reaction, the 5'-noncoding regions of GBV-C/HGV and HCV were amplified from plasma or sera of 209 individuals infected with human immunodeficiency virus type 1 (HIV-1). As verified by Southern blot analysis, GBV-C/HGV and HCV infection were detected in 37 (17.7%) and 22 (10.5%) of 209 HIV-1-infected individuals, respectively. GBV-C/HGV infection was significantly associated with homosexual sex (P = 0.044) and was more common than HCV infection among HIV-1-infected homosexual men (P = 0.006). The prevalence of GBV-C/HGV infection was nearly equal in women infected with HIV-1 via high-risk heterosexual sex (14.0%) or injection drug use (IDU) (17.5%). By contrast, HCV infection was associated significantly with women reporting IDU when compared to women reporting high-risk heterosexual sex (P < 0.0001). Alanine aminotransferase levels were elevated in HIV-1-infected individuals who were co-infected with HCV (P = 0.009), but not with GBV-C/HGV (P = 0.9). The high prevalence of GBV-C/HGV infection in HIV-1-infected nondrug-injecting homosexual men and among women engaging in high-risk heterosexual sex is consistent with transmission by the mucosal route and with acquisition of infection by the receptive rather than insertive partner.

Complete nef gene sequence of HIV type 1 subtype B' from professional plasma donors in the People's Republic of China.

PIP: Probably more than 150,000 people are infected with HIV in China. Although HIV-1 infection was initially limited to the southern province of Yunnan, it is increasingly spreading in the interior of the country, and multiple HIV-1 subtypes have been identified, including B, C, and E. Envelope (env) gene sequences of HIV-1 strains from Yunnan resemble HIV subtype B' strains from Thailand. The authors analyzed the nef gene from HIV-1-infected professional plasma donors to determine if deletions and/or insertions are present in the nef gene of HIV-1 strains from China. Blood was collected from 3 men of mean age 42 years and 4 women of mean age 40 between December 1996 and April 1997 in eastern China, blotted onto neonatal 903 filter paper and allowed to dry, then analyzed. Sequence alignment and comparison of the gene found that the HIV-1 strains from China diverged from well-characterized subtype B strains from North America, Thailand, and Papua New Guinea by 9.6-16.7% and 8-20% at the nucleotide and deduced amino acid levels, respectively. On the basis of sequence and phylogenetic analyses of a 550-nucleotide region spanning the main neutralizing domain, the V3 loop, of the gp120-encoding env gene, all of the 7 strains from China were more similar to the subtype B' HIV of Thailand than to the subtype B of North America.^ieng

Gradual loss of IgG antibodies against GB virus C/hepatitis G virus in a patient with AIDS.

GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense, single-stranded RNA virus belonging to the family Flaviviridae and is distantly related to hepatitis C virus (HCV). GBV-C/HGV can be transmitted by the parenteral and the sexual route. Among individuals infected with human immunodeficiency virus type 1 (HIV-1) by the sexual route, we and others have demonstrated a high prevalence of GBV-C/HGV infection. Recently, Woolley and colleagues reported that AIDS patients co-infected with GBV-C/HGV had a significantly lower mean CD4 cell count than AIDS patients without GBV-C/HGV infection, suggesting that GBV-C/HGV antibody may be lost with progression to AIDS. To our knowledge no data are available on the loss of antibody against GBV-C/HGV in AIDS patients. We now report on an HIV-infected patient who exhibited gradual loss of IgG antibodies against GBV-C/HGV, as well as HCV, with progression of HIV disease.

Sequence and phylogenetic analyses of HIV-1 infection in Vietnam: subtype E in commercial sex workers (CSW) and injection drug users (IDU).

More than 4,000 persons with human immunodeficiency virus type 1 (HIV-1) infection have been identified in Vietnam through sentinel surveillance since 1990, when the first case of HIV-1 infection was diagnosed in a young woman in Ho Chi Minh City. Currently, the estimated HIV-1 seroprevalences of 10% for injection drug users (IDU) and 3% for female commercial sex workers (CSW) in Vietnam are comparable to those observed in the same risk groups in Thailand five years ago. To clarify if concurrent epidemics with different HIV-1 subtypes (or clades) are occurring among different high-risk behavior groups in Vietnam, we conducted a genotypic analysis of HIV-1 by amplifying and sequencing a 325-nucleotide region spanning the principal neutralizing domain, or V3 loop, of the gp120-encoding env gene from genomic DNA extracted from dried, filter paper-blotted blood samples, collected in April/May and August/September 1995 from 8 HIV-1-seropositive CSW in Ho Chi Minh City, Can Tho and An Giang provinces and from 16 IDU in Ho Chi Minh City, Hanoi, Nha Trang and An Giang province. Sequence alignment and comparison with other HIV-1 subtypes indicated that the HIV-1 strains from CSW and IDU in Vietnam were genetically most similar to subtype E strains from Cambodia. The interstrain genetic variation among the Vietnam HIV-1 env sequences ranged from 0.3% to 9.0% (mean, 4.6%). Phylogenetic analysis verified that some of the Vietnam HIV-1 strains formed discrete clusters and were indistinguishable from other Southeast Asian strains. The demonstration of subtype E in both CSW and IDU in Vietnam contrasts sharply with the previously observed HIV-1 clade restriction in these high-risk behavior groups in nearby Thailand.

HIV type 1 subtype E in commercial sex workers and injection drug users in southern Vietnam.

PIP: Nationwide HIV-1 seroprevalence rates in Vietnam are estimated to be almost 10% for IV drug users (IVDUs), 3% for female prostitutes, and 2% for males attending clinics for sexually transmitted diseases. These estimated prevalences are comparable to those observed in the same risk groups in Thailand 5 years ago. Blood samples were analyzed from two female HIV-1-seropositive prostitutes and three male IVDUs in southern Vietnam during April and May 1995. HIV-1 infection was confirmed by nested PCR in all five samples. Sequence alignment and comparison of the 325-nucleotide region with the major HIV-1 subtypes from widely separated geographic regions indicate that the Vietnam HIV-1 strains are genetically most similar to virus strains from Thailand, diverging from well-characterized subtype E strains by 3.1-5.9% and 5.6-12.0% at the nucleotide and deduced amino acid levels, respectively. The interstrain genetic variation among the Vietnam env sequences was 2.5-4.9%. None of the prostitutes and IVDUs studied had traveled to or worked in Thailand or Cambodia, and neither of the prostitutes used IV drugs, suggesting that they were infected sexually with indigenous strains circulating within Vietnam. The phylogenetic clustering of the Vietnam HIV-1 strains and their relative low degree of sequence variability are consistent with a founder effect and the recent introduction of HIV-1 subtype E.^ieng

Mechanisms of the in vitro antimutagenic action of chlorophyllin against benzo[a]pyrene: studies of enzyme inhibition, molecular complex formation and degradation of the ultimate carcinogen.

Mechanisms of the antimutagenic action of chlorophyllin (CHL) towards benzo[a]pyrene (BP) were studied in vitro. In the Salmonella assay, CHL inhibited the mutagenic activity of BP in the presence of an S9 activation system and was particularly effective against the direct-acting ultimate carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Spectral studies indicated that the time-dependent hydrolysis of BPDE to tetrols was augmented in the presence of CHL concentrations on the order of 5 microM. Dose-related inhibition of several cytochrome P450-dependent enzyme activities was observed upon addition of CHL to in vitro incubations. Spectral changes for the interaction between CHL and cytochrome P450 indicated that CHL does not bind to the active site of the enzyme, but exerts its inhibitory effect indirectly. This was achieved by inhibiting NADPH-cytochrome P450 reductase (Ki approximately 120 microM with cytochrome c as substrate), and did not involve lowering of the effective substrate concentration by complex formation with the procarcinogen. It is concluded that the in vitro antimutagenic activity of CHL towards BP involves accelerated degradation of the ultimate carcinogen, with inhibition of carcinogen activation occurring only at high CHL concentrations. The latter mechanism is unlikely to occur in vivo following p.o. administration due to the limited uptake of CHL from the gut, but tissue concentrations may be sufficiently high to cause degradation of BPDE.