Multiple human beta interferon genes.

Analysis of human beta interferon (IFN) mRNA preparations obtained from poly(I) . poly (C)-induced human diploid fibroblasts (FS-4) and from several similarly induced human-mouse somatic cell hybrids by electrophoresis through agarose-CH3HgOH tube gels led to the detection of at least five translationally active human IFN-beta mRNA species. The results obtained are consistent with the existence of IFN-beta genes on different human chromosomes. Marked cell-dependent variability in the expression of these IFN mRNA species was observed.

Chromosomal location of a human alpha interferon gene family.

To determine the chromosomal location of the human alpha interferon genes, we scored a series of human/rodent somatic cell hybrids for the presence of DNA sequences hybridizing to an alpha 1 interferon DNA probe. The presence of human chromosome 9 in a hybrid correlated with the presence of a family of alpha interferon genes.

Assignment of the murine interferon sensitivity and cytoplasmic superoxide dismutase genes to chromosome 16.

Both hybrids of mouse and human microcells and whole cell hybrids generated by the fusion of primary mouse cells and SV40-transformed human fibroblasts were used to establish the syntenic association of the murine cytoplasmic superoxide dismutase and the interferon sensitivity genes on mouse chromosome 16. This assignment adds two new markers to chromosome 16 and provides another example of an evolutionarily conserved linkage. This finding also provides an animal model both for cellular responsiveness to interferon and for Down's syndrome.

Interferon-beta-related DNA is dispersed in the human genome.

Interferon-beta 1 (IFN-beta 1) complementary DNA was used as a hybridization probe to isolate human genomic DNA clones lambda B3 and lambda B4 from a human genomic DNA library. Blot-hybridization procedures and partial nucleotide sequencing revealed that lambda B3 is related to IFN-beta 1 (and more distantly to IFN-alpha 1). Analyses of DNA obtained from a panel of human-rodent somatic cell hybrids that were probed with DNA derived from lambda B3 showed that lambda B3 is on human chromosome 2. Similar experiments indicated that lambda B4 is not on human chromosomes 2, 5, or 9. The finding that DNA related to the IFN-beta 1 gene (and IFN-alpha 1 gene) is dispersed in the human genome raises new questions about the origins of the interferon genes.

Human beta interferon gene localization and expression in somatic cell hybrids.

The human fibroblast interferon gene beta 1 was mapped to human chromosome 9. Sequence homology with a beta 1 cDNA clone was detected in both genomic DNA and induced mRNA of human/mouse or human/hamster somatic cell hybrids containing human chromosome 9, but not in lines lacking this chromosome or those retaining a complex translocation involving chromosomes 9 and 11. Interferon mRNA that did not share sequence homology with the beta 1 cDNA clone was detected in lines containing human chromosomes 2 and 5 but lacking chromosome 9, suggesting the presence of other unlinked interferon sequences in the human genome.

Effect of exposure to calcium entry blockers on doxorubicin accumulation and cytotoxicity in multidrug-resistant cells.

Flow cytometry has been used to measure doxorubicin (DOX) retention in several pairs of drug-sensitive and multidrug-resistant (MDR) cell lines and in unselected human tumor cell lines. Co-exposure to several agents that have been reported to reverse multidrug resistance, particularly calcium entry blockers (CEBs), produced a dose-dependent increase in DOX accumulation in MDR cell lines. In MDR Chinese hamster ovary cells (CHRC5), DOX levels declined rapidly following removal of CEBs, reaching a plateau value above that found in cells treated with DOX alone; this small increase probably represents DOX that is not accessible to the p170 efflux pump overexpressed in these cells. Increased DOX retention could be observed even after brief exposure to CEBs and washout and correlates with a decrease in cell proliferation over a 3-day growth assay. These results suggest that only a brief inhibition of drug efflux is sufficient to produce a meaningful reversal of drug resistance.

Effect of verapamil on doxorubicin cardiotoxicity: altered muscle gene expression in cultured neonatal rat cardiomyocytes.

Verapamil reverses multidrug resistance acquired by cancer cells during treatment with chemotherapeutic agents such as doxorubicin by inhibiting the function of P-glycoprotein. Verapamil has also been suggested to potentiate the cardiotoxicity of doxorubicin. We have recently demonstrated that selective inhibition of cardiac muscle gene expression is among the earliest events in doxorubicin cardiotoxicity. To explore the influence of verapamil on doxorubicin cardiotoxicity, we evaluated [14C]-doxorubicin accumulation, cardiac muscle gene expression by Northern blot analysis, and ultrastructural changes in cultured cardiomyocytes in the presence and absence of verapamil. Treatment with a combination of doxorubicin and verapamil for 24 h did not augment doxorubicin accumulation in cardiomyocytes, although substantial augmentation of doxorubicin accumulation by verapamil in cardiac fibroblasts was observed. Further, treatment with verapamil for 24 h did not augment the decrease in expression of muscle genes induced by doxorubicin (myosin light chain 2 slow, troponin I, M isoform creatine kinase). However, we found that verapamil reduced alpha-actin gene expression in a direct, doxorubicin-independent manner. Furthermore, the effect of doxorubicin plus verapamil on alpha-actin gene expression was additive over a wide range of doxorubicin and verapamil concentrations, resulting in a selective augmentation of doxorubicin-induced inhibition of gene expression for this single muscle protein gene. This was reflected in a substantial increase in cardiac myocyte damage when treatment with verapamil and doxorubicin was compared to treatment with doxorubicin alone by thin section electron microscopy. This suggests a possible mechanism by which verapamil may potentiate doxorubicin cardiotoxicity.

Intracellular expression of P-glycoprotein in a human colon tumor cell line.

A number of human tumor cell lines originating in tissues which normally express high levels of P-glycoprotein were examined for the expression of mdr-1 RNA and P-glycoprotein and their sensitivity to doxorubicin and vincristine. There was a wide variation in expression levels and in drug sensitivity among the cell lines. In the human colon tumor cell line, LS 174T, high levels of P-glycoprotein and mdr-1 RNA were observed, but this line was very sensitive to doxorubicin and vincristine. Immunoprecipitation of P-glycoprotein from preparations of membrane and cytoplasmic proteins and immunofluorescence studies using anti-P-glycoprotein antibodies revealed that P-glycoprotein in these cells is not associated with the plasma membrane, but is, instead, found intracellularly. These results emphasize the need for functional analysis of P-glycoprotein in cells that express this mediator of multidrug resistance.

RS-33295-198: a novel, potent modulator of P-glycoprotein-mediated multidrug resistance.

A novel multidrug resistance modulator, RS-33295-198, circumvented drug resistance in human, mouse, and Chinese hamster cell lines overexpressing P-glycoprotein. It enhanced the antiproliferative activity of doxorubicin, vincristine, etoposide, and paclitaxel and increased doxorubicin retention in multidrug-resistant hamster CHRC5 cells. RS-33295-198 modulated doxorubicin resistance in a murine P388/ADR leukemia model when administered ip via continuous minipump delivery, ip by bolus injection, and orally; it also improved the efficacy of vincristine toward P388/VCR leukemia when given ip or po. RS-33295-198 showed weak activity in enhancing doxorubicin efficacy against a multidrug-resistant human sarcoma xenograft.

Modulation of doxorubicin efficacy in P388 leukemia following co-administration of verapamil in mini-osmotic pumps.

Co-administration of doxorubicin and verapamil in Alzet mini-osmotic pumps increased the survival of B6D2F1 mice bearing the multidrug-resistant P388/ADR leukemia. A range of doxorubicin and verapamil combinations was studied to define dose-dependent efficacy and toxicity. High doses of doxorubicin (10 mg/kg/day) and verapamil (150 mg/kg/day) could be administered alone without any effect on survival. However, combining high doses of these two agents resulted in host toxicity. Doxorubicin doses of 1 to 10 mg/kg/day in combination with verapamil at 25-100 mg/kg/day were found to improve survival compared with either agent alone. Combination therapy also improved the survival of mice bearing the drug-sensitive P388/0 leukemia when compared to anthracycline treatment alone. The efficacy of the mini-osmotic pump delivery protocol was compared with other regimens delivering the same total cumulative dose of doxorubicin via repeated i.p. injections.

Fibroblast interferon in man is coded by two loci on separate chromosomes.

We have examined viral and poly(rl):poly(rC) induction of interferon synthesis in several human, mouse and Chinese hamster cell lines, and in hybrids derived from the fusion of such cells. We observed species and cell-type differences in inducer effectiveness and in the kinetics of interferon production. In some cases, parental characteristics are preserved in somatic cell hybrids, and in other cases, the expression of the donor phenotype is modulated by the epigenetic state of the recipient cell. Mapping studies in human/mouse and human/Chinese hamster hybrids indicate that there are at least two structural genes for human fibroblast interferon. Chromosomes 2 and 5 each contain genetic information for the synthesis of fibroblast interferon. Gene dosage experiments indicate that one gene is on the long arm of chromosome 2 and another is on the short arm of chromosome 5. Leukocyte interferon genes could not be mapped to these chromosomes, but this negative result could be influenced by the epigenetic state of the hybrid cells.

S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells.

We investigated the cell cycle modulation of dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase, 7,8-dihydroxyfolate:NADP+ oxidoreductase, EC 1.5.1.3) levels in methotrexate-resistant Chinese hamster ovary cells synchronized by mitotic selection. DNA content and DHFR concentration were analyzed throughout the cell cycle by standard biochemical techniques and by double fluorescence staining utilizing the fluorescence-activated cell sorter. We found an S phase-specific period of DHFR biosynthetic activity. Commencing within hour 2 of S phase and continuing throughout the duration of S phase, there is a 90% increase in DHFR specific activity. This results from an approximately 2.5-fold increase in the level of DHFR, while total soluble protein increases 50% during the same period. This increase is the result of new synthesis of DHFR molecules initiated after the cell is physiologically committed to DNA replication. This increase in DHFR activity through S phage parallels the increasing rate of [3H]thymidine incorporation during the same interval. The maximum peak of DHFR activity is coincident with the maximum rate of DNA synthesis, both activities occurring during the bulk of DNA replication within the last stages of the 6.5-hr S phase.

Multifactorial resistance in LS174T human colon carcinoma cells selected with doxorubicin.

A series of doxorubicin-resistant variants of the human LS174T colon carcinoma cell line was generated by stepwise selection. These variants also exhibited increased resistance to vinblastine, etoposide, cis-platinum, and melphalan, suggesting that resistance was multifactorial. The parental LS174T cell line and 3 resistant variants were examined for over-expression of P-glycoprotein, changes in total cellular glutathione content, and the level of topoisomerase-II expression. Changes in all of these parameters were observed in the doxorubicin-selectants, along with a marked shift in the intracellular distribution of doxorubicin. P-glycoprotein RNA and protein levels were increased 2- to 3-fold in the resistant variants, while total glutathione levels increased 1.4- to 2.1-fold. Treatment with DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione biosynthesis, was able to reverse resistance to cis-platinum and melphalan in these variants, but had little effect on doxorubicin resistance. Immunoblot analysis of cell extracts indicated that the level of DNA topoisomerase II (EC 5.99.1.3) in the doxorubicin-resistant LS174T cells was decreased by approximately 50% compared with the parental cell line. Doxorubicin was mainly localized to the cytoplasm in resistant cells, while in the parent line it was mostly found in the nucleus. This constellation of changes suggests that selection with doxorubicin activated several mechanisms of resistance involving drug transport, metabolism, and ability to reach nuclear target sites.

Isolation and characterization of doxorubicin-resistant Lewis lung carcinoma variants.

A series of drug-resistant variants of the murine Lewis lung carcinoma (3LL-CK) cell line has been isolated using stepwise selection in increasing concentrations of doxorubicin. These variants exhibited classical multidrug resistance as evidenced by decreased doxorubicin accumulation, cross-resistance to vinblastine, reversibility of resistance by verapamil, and overexpression of P-glycoprotein. When the doxorubicin-resistant 3LL-CK cell populations were injected into the tail veins of B6D2F1 mice, their metastatic abilities were consistently reduced compared with that of the parental line.