Biomedical subjects

H Fan

Publications and source records attributed to H Fan.

238 records · Page 14Linked to original sources

PubMedDate not supplied

Purification of Moloney murine leukemia virus chromatin from infected cells by an affinity method.

Our goal was to develop a system to study proteins that associate in vivo with the Moloney murine leukemia virus (M-MuLV) enhancer elements by the isolation of intact proviral chromatin. The M-MuLV long terminal repeats (LTRs) contain tandemly repeated transcriptional enhancer sequences consisting of smaller motifs that bind cellular DNA-binding proteins implicated in transcriptional regulation. The M-MuLV enhancers are also important for disease specificity and latency of disease induction. To enrich for proviral chromatin containing M-MuLV LTR sequences, an affinity purification scheme was employed that relies on the affinity of bacterial Lac repressor protein for Lac operator (LacO) DNA sequences. An infectious M-MuLV recombinant was constructed that contains bacterial LacO sequences inserted into a nonessential region downstream from the 5' LTR of the virus (M-MuLV-LacO). Nuclei from M-MuLV-LacO-infected cells were digested with PvuII (which will liberate an LTR fragment containing LacO sequences), and digested chromatin was leached from the nuclei in hypotonic buffer. M-MuLV-LacO chromatin was then recovered by binding to an affinity matrix consisting of a beta-galactosidase-Lac repressor fusion protein anchored to acrylamide beads by an anti-beta-galactosidase monoclonal antibody [7]. Specifically bound chromatin was eluted under physiological conditions by incubation with the galactose analog isopropyl-beta-D-thiogalactopyranoside. Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin by this method.

Animals↗

PubMedDate not supplied

Non-function of a Moloney murine leukaemia virus regulatory sequence in F9 embryonal carcinoma cells.

Moloney murine leukaemia virus (M-MuLV) infection of embryonal carcinoma (EC) cells results in the integration of proviral DNA into the host cell genome, but not in virus production. One suggested explanation for the lack of viral gene expression in EC cells has been methylation of the integrated viral DNA. However, subsequent reports indicated that integration of the M-MuLV DNA occurs soon after infection, but that viral DNA methylation occurs considerably later. Nevertheless, viral gene expression is not observed even at early times. One possible explanation is that certain M-MuLV regulatory sequences do not function in EC cells. We now present evidence which supports this hypothesis.

Acetyltransferases↗

PubMedDate not supplied

Suppression of leukaemia virus pathogenicity by polyoma virus enhancers.

The long terminal repeats (LTRs) of retroviruses contain sequences necessary for the initiation and termination of retroviral transcription. These sequences include promoter elements, transcriptional termination signals and transcriptional enhancer elements. The enhancer elements of Moloney murine leukaemia virus (M-MuLV) are localized in a tandemly repeated region (approximately 75 base pairs (bp) long), which lies 5' to the CAT and TATA promoter elements in the U3 region of the LTR (see Fig. 1). We have shown that the tandem repeats are required both for LTR promoter activity, as measured by transient expression assays, and for biological activity, as measured by production of infectious virus. Furthermore, they can be replaced by transcriptional enhancers from the F101 host-range mutant of polyoma virus without loss of function. We report here that the addition of the polyoma (PyF101) enhancers to the M-MuLV LTRs (either with or without the M-MuLV tandem repeats) results in complete loss of viral leukaemogenicity, even though the virus can replicate to high titres in tissue culture fibroblasts and can establish infection in animals.

Animals↗

PubMedDate not supplied

Evaluation of D-methionine as a cytoprotectant in cisplatin treatment of an animal model for ovarian cancer.

BACKGROUND: To evaluate the use of D-methionine(D-met) as a cytoprotectant in the context of clinically relevant doses of cisplatin. MATERIALS AND METHODS: Forty five Fischer rats were injected intraperitoneally with 10(6) NuTu-19 cells and treated as follows: group 1 was the control group and received no treatment, group 2 received cisplatin 4 mg/kg and group 3 received cisplatin 4 mg/kg plus D-met. There were two groups that received high dose cisplatin. Group 4 received cisplatin 8 mg/kg and group 5 received cisplatin 8 mg/kg plus D-met. Treatment was initiated four weeks after injection of the NuTu-19 cells, and consisted of four weekly intraperitoneal injections. Serum BUN and creatinine levels in the high dose groups evaluated nephrotoxicity and clinical outcome was measured by mean survival using Kaplan Meier analysis. RESULTS: There were no significant elevations in serum BUN or creatinine levels in any of the rats treated with high dose cisplatin. In the animals given cisplatin 8 mg/kg plus D-met, death from toxicity was prevented and all animals completed four treatments. In contrast, only two animals in group 4 (cisplatin 8 mg/kg alone) completed 4 treatments. There was a significant improvement in survival for the animals given D-met. (p = .0001) In all treated groups except for group 4, there was an improvement in survival compared to the control group. When comparing groups 2 and 3 (4 mg/kg +/- D-met), there was a subjective decrease in tumor response for group 3 but mean survival was not statistically different. (91 vs. 81 days; p = 0.07) A comparison of groups 2 and 5 revealed no survival benefit using high dose cisplatin with D-met. (91 vs. 79 days; p = 0.10). CONCLUSIONS: Our results indicate that D-methionine provides cytoprotection against cisplatin toxicity without significant compromise of antitumor activity. All though D-methionine allowed for significant dose intensification of cisplatin above standard doses, there was no survival advantage noted in this group of animals. The indications for its use in the treatment of ovarian cancer remain to be determined.

Animals↗