Cooperative therapeutic effects of androgen ablation and adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy in experimental prostate cancer.
(9/2453)
Adenovirus-mediated transduction of the herpes simplex thymidine kinase gene (HSV-tk) in conjunction with ganciclovir (GCV) has been shown to result in significant growth suppression and to enhance survival in a model of mouse prostate cancer. However, this therapeutic activity is not sustained, because in most cases tumors eventually regrow and ultimately cause the death of the host. Androgen ablation, an inducer of apoptosis in prostate cells which is used widely as palliative therapy in patients with prostate cancer, was combined with HSV-tk plus GCV using an androgen-sensitive mouse prostate cancer cell line. The combination of castration and HSV-tk plus GCV led to markedly enhanced tumor growth suppression in both subcutaneous and orthotopic models compared with either treatment alone and resulted in an enhanced survival in which combination-treated animals lived twice as long as controls in the subcutaneous model and over 50% longer than controls in the orthotopic model. Further analysis of apoptotic activity demonstrated high levels of apoptosis only in combined androgen ablation and HSV-tk plus GCV-treated tumors after 14 days of growth in an androgen-depleted environment and 8 days after HSV-tk plus GCV therapy. At this time, the apoptotic index, but not the percent of necrotic tissue, was significantly higher for combination therapy-treated tumors relative to control-treated tumors or either treatment alone. These data indicate that the therapeutic effects of androgen ablation and HSV-tk plus GCV are cooperative and that increased apoptosis may, in part, underlie these activities. (+info)
Functional domains of c-myc promoter binding protein 1 involved in transcriptional repression and cell growth regulation.
(10/2453)
We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-myc promoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation. (+info)
Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy.
(11/2453)
Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway. (+info)
Suppression of intrachromosomal gene conversion in mammalian cells by small degrees of sequence divergence.
(12/2453)
Pairs of closely linked defective herpes simplex virus (HSV) thymidine kinase (tk) gene sequences exhibiting various nucleotide heterologies were introduced into the genome of mouse Ltk- cells. Recombination events were recovered by selecting for the correction of a 16-bp insertion mutation in one of the tk sequences. We had previously shown that when two tk sequences shared a region of 232 bp of homology, interruption of the homology by two single nucleotide heterologies placed 19 bp apart reduced recombination nearly 20-fold. We now report that either one of the nucleotide heterologies alone reduces recombination only about 2.5-fold, indicating that the original pair of single nucleotide heterologies acted synergistically to inhibit recombination. We tested a variety of pairs of single nucleotide heterologies and determined that they reduced recombination from 7- to 175-fold. Substrates potentially leading to G-G or C-C mispairs in presumptive heteroduplex DNA (hDNA) intermediates displayed a particularly low rate of recombination. Additional experiments suggested that increased sequence divergence causes a shortening of gene conversion tracts. Collectively, our results suggest that suppression of recombination between diverged sequences is mediated via processing of a mispaired hDNA intermediate. (+info)
Identification of the canarypox virus thymidine kinase gene and insertion of foreign genes.
(13/2453)
We mapped the canarypox virus (CaPV) thymidine kinase (TK) gene within a 5.8-kbp XbaI fragment of the genome by Southern blotting using the fowlpox virus (FPV) TK gene as a probe. Nucleotide sequence analysis of the fragment revealed seven open reading frames (ORFs) showing gene organization similar to that of FPV. The TK gene contained in this region had an ORF of 179 amino acids encoding a polypeptide with a putative molecular mass of 20.0 kDa. An A/T-rich region and a transcription termination signal, TTTTTAT, were found upstream and at the end of the ORF, which is consistent with poxvirus early gene regulation. The consensus sequence of the late promoter TAAAT also overlapped with the initiation codon of the ORF. The amino acid sequence similarity between the TK genes of CaPV and FPV, avipoxviruses, was 64.2%, which was lower than the similarities between vaccinia and variola orthopoxviruses (97.2%) and between Shope fibroma and myxoma leporipoxviruses (82.6%). However, the monophyly of avian clades of CaPV and FPV was supported by phylogenetic analysis. We then inserted the genes encoding lacZ, luciferase (luci), and envelope of human T-lymphotropic virus type 1 (HTLV-1 env) into the TK gene of CaPV to evaluate its suitability as an expression vector. The recombinant viruses obtained were unstable, although the foreign genes were expressed efficiently in the mammalian cells infected with the viruses. (+info)
Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer.
(14/2453)
The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents. (+info)
The mismatch repair protein, hMLH1, mediates 5-substituted halogenated thymidine analogue cytotoxicity, DNA incorporation, and radiosensitization in human colon cancer cells.
(15/2453)
Deficiency in DNA mismatch repair (MMR) is found in some hereditary (hereditary nonpolyposis colorectal cancer) and sporadic colon cancers as well as other common solid cancers. MMR deficiency has recently been shown to impart cellular resistance to multiple chemical agents, many of which are commonly used in cancer chemotherapy. It is therefore of interest to find an approach that selectively targets cells that have lost the ability to perform MMR. In this study, we examine the response of MMR-proficient (hMLH1+) and MMR-deficient (hMLH1-) colon carcinoma cell lines to the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) before and after irradiation. These dThd analogues are used clinically as experimental sensitizing agents in radioresistant human cancers, and there is a direct correlation between the levels of dThd analogue DNA incorporation and tumor radiosensitization. In contrast to the well-characterized, marked increase in cytotoxicity (> 1 log cell kill) found with 6-thioguanine exposures in HCT116/3-6 (hMLH1+) cells compared to HCT116 (hMLH1-) cells, we found only modest cytotoxicity (10-20% cell kill) in both cell lines when treated with IdUrd or BrdUrd for 1 population doubling. Upon further analysis, the levels of halogenated dThd analogues in DNA were significantly lower (two to three times lower) in HCT116/3-6 cells than in HCT116 cells, and similar results were found in Mlh1+/+ spontaneously immortalized murine embryonic fibroblasts and fibroblasts from Mlh1 knockout mice. As a result of the higher levels of the dThd analogue in DNA, there was an increase in radiation sensitivity in HCT116 cells but not in HCT116/3-6 cells after pretreatment with IdUrd or BrdUrd when compared to treatment with radiation alone. Additionally, we found no differences in the cellular metabolic pathways for dThd analogue DNA incorporation because the enzyme activities of dThd kinase and thymidylate synthase, as well as the levels of triphosphate pools, were similar in HCT116 and HCT116/3-6 cells. These data suggest that the hMLH1 protein may participate in the recognition and subsequent removal of halogenated dThd analogues from DNA. Consequently, whereas MMR-deficient cells and tumor xenografts have shown intrinsic resistance to a large number of chemotherapeutic agents, the 5-halogenated dThd analogues appear to selectively target such cells for potential enhanced radiation sensitivity. (+info)
Role of the individual interferon systems and specific immunity in mice in controlling systemic dissemination of attenuated pseudorabies virus infection.
(16/2453)
The importance of each of the two interferon (IFN) systems in impeding herpesvirus replication and in stimulating virus-specific lymphocytes to control an acute systemic infection is not completely understood. To further our knowledge, pseudorabies virus, attenuated by deletion of the glycoprotein E gene to impair its neurovirulence and by deletion of the thymidine kinase gene (gE-TK-PRV), was used to infect wild-type 129Sv/Ev and congenic mice with immune system-associated genetic deficiencies. Mice with mature B and T lymphocytes but lacking either one or both functional receptors for members of each of the two IFN families were infected with gE-TK-PRV. At 3 and 7 but not 14 days after infection, replicating gE-TK-PRV could be isolated only from livers or spleens of mice lacking the receptors for both IFN families, and these mice survived the infection. Therefore, functional IFN receptors were not required to induce a protective immune response against an acute infection with gE-TK-PRV. Furthermore, PRV-specific antibodies of all immunoglobulin G isotypes were produced in these mice. Mice without mature B and T lymphocytes and lacking either one or both functional receptors for members of each of the two IFN families were also infected with gE-TK-PRV. Three days after infection, replicating virus could be isolated only from mice lacking both mature B and T lymphocytes and functional IFN receptors, and these mice were not able to clear the virus. We present evidence that mice with an intact gamma IFN system but without mature B and T cells were able to prevent systemic dissemination of gE-TK-PRV. (+info)