We report that treatment of 2.2.15, a human hepatoblastoma-derived cell line in which hepatitis B virus is actively replicating, with the carbocyclic analogue of 2'-deoxyguanosine [Shealy, Y. F., O'Dell, C. A., Shannon, W. M. & Arnett, G. (1984) J. Med. Chem. 27, 1416-1421] resulted in the nearly complete cessation of viral replication, as monitored by the absence of both intracellular episomal and secreted viral DNAs and by the absence of viral DNA polymerase activity. The drug was nontoxic in concentrations up to 200 times the minimum effective inhibitory concentration.
The frequency of sister chromatid exchanges (SCE) in vivo and chromosome aberrations and/or alterations were analyzed from the bone marrow cells of the treated dbrB tumor-bearing DBA/1J inbred mouse host. The results were compared with analogous data obtained from the bone marrow cells of untreated tumor-bearing mice for evaluation of the "indirect," i.e., somatic stress, effect on the normal host cells following triple-agent therapy intended for a mammary adenocarcinoma. Misonidazole (MIS), which is a known radiosensitizing drug, microwave hyperthermia (delta), and X-radiation (X) were used as therapeutic agents. Significant (P less than 0.05) numbers of SCE were induced in the bone marrow cells of the mice whose tumors received these triple-agent treatments (MIS + delta + X) simultaneously as compared with values of SCE per cell noted in bone marrow cells of untreated tumor-bearing control mice. The highest number of chromosome aberrations and alterations, including an increase in heteroploidy, was also noticed in the bone marrow cells of the mice whose tumors were treated simultaneously with MIS + delta + X. The triple-agent therapy on dbrB tumor also resulted in an unusually high polyploid metaphase plate in the bone marrow cell consisting of 320 chromosomes, indicating that this mode of therapy may act directly on the genetic material of the tumor-bearing host cells, inducing cytogenetic abnormalities as a side effect.
A rapidly proliferating mammary adenocarcinoma designated dbrB growing s.c. in isogeneic female DBA/1J mice in their 278th passage of transfer constituted the experimental host tumor system. A 5-bromodeoxyuridine pellet (2.5 mg/g body weight) was used for sister chromatid exchange analysis of the 0.5-cu cm dbrB tumor. Experiments were performed on Group I, untreated tumor-bearing mice, and Group II, tumor-bearing mice treated with triple agents: misonidazole (1 mg/g body weight); 42.5 degrees microwave hyperthermia for 10 min; and X-rays administered singly or in combination at about 4 hr following the implantation of a 5-bromodeoxyuridine pellet. The X-ray treatments consisted of 400, 1000, 1500, and 2000 rads, respectively. X-rays and hyperthermia were delivered to the tumors directly, while the rest of the mouse body was lead shielded. Survival of the untreated tumor-bearing control mice in Group I was 12 +/- 3 (S.D.) days, whereas the mice in Group II whose tumors were treated with misonidazole, hyperthermia, and 2000 rads of X-rays survived 27 +/- 3 days. 5-bromodeoxyuridine per se had no effect on the survival of the experimental mice. Only a dose of 400 rads administered to the dbrB tumors permitted detailed evaluation of chromosomal analyses, whereas the larger doses of radiation caused cellular destruction. Simultaneous treatment with triple agents resulted in sister chromatid exchanges of 33.78 +/- 0.39/cell as compared with sister chromatid exchanges of 14.74 +/- 0.39/cell of untreated control tumors. This mode of treatment also induced various types of chromosomal abnormalities in the tumor cells.
This study revealed that the triangular shaped spleen of X/Gf mice constitutes a genetically dominant characteristic over normal spleen shape of C3H mice. The agouti coat color of C3H mice is dominant over the white color of the X/Gf strain. The mode of transmission of triangular shape of the spleen in X/Gf mice, as well as their white coat color indicates Mendelian autosomal inheritance involving two independent genes. Although the pedigree lends support to this inference, we are aware that the small number of F2 offspring available for this study limits the conclusions to be drawn from the segregation ratio and statistical evaluation.
The proliferation and genetic characteristics of a mouse mammary adenocarcinoma designated dbrB are described. This tumor was maintained as subcutaneous serial transplants in syngeneic inbred mice of the DBA/1J strain. Quantitative in vivo sister chromatid exchange(s) (SCE) were determined in the tumor cells and in the bone marrow cells of the host strain. Over a twofold increase in SCE, frequency was noted in the dbrB tumor cells compared to the bone marrow cells. The high frequency of SCE in the dbrB tumor was assumed to be caused by the high degree of malignancy of this tumor. Such information indicating genetic stress may serve for evaluation of the degree of malignancy in neoplasms.
We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.
We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.
Chloroquine and its analogue hydroxychloroquine (HCQ) have been shown to inhibit a variety of viral infections including influenza and adenovirus through blockade of viral entry via inhibition of endosomal acidification. We have extended these observations to human immunodeficiency virus type 1 (HIV-1) infection utilizing primary T cells and monocytes, a T cell line (CEM), and a monocytic cell line (U-937). HCQ inhibited HIV-1 replication (>75%), as measured by reverse transcriptase activity, in the primary T cells and monocytes as well as the T cell and monocytic cell lines. HCQ itself had no anti-reverse transcriptase activity and was not toxic to the cells at concentrations inhibitory to viral replication. Intracytoplasmic staining with an anti-p24 antibody, 24 h after infection, revealed the presence of intracytoplasmic virus, suggesting that the drug does not block viral entry. The production of steady-state HIV-1 mRNA was not affected by HCQ in that comparable levels of HIV-1 mRNA could be detected by Northern blot analysis and by in situ hybridization in both the HCQ-treated and untreated cells. However, HCQ does appear to affect production of infectious HIV-1 virions because viral isolates from HCQ-treated cells could not infect target CEM cells. These data suggest that HCQ may be useful adjunctive therapy in the treatment of HIV-1 infection.
