Increased expression and DNA-binding activity of transcription factor Sp1 in doxorubicin-resistant HL-60 leukemia cells.

The processes responsible for the multidrug-resistant (Mdr) phenotype in Adriamycin (doxorubicin)-resistant HL-60 leukemia cells (HL-60/AR) are not defined. Since enhanced transcription of resistance-related proteins is associated with Mdr cells, we sought to determine whether changes in the expression of specific transcription factors were a feature characteristic of the Mdr process. Nuclear extracts were prepared from wild-type and resistant cells and compared for their ability to bind DNA consensus sequences for the transcription factors Sp1 and NF kappa B contained in the 5' long terminal repeat region of human immunodeficiency virus type 1. Southwestern (DNA-protein) blots showed a family of DNA-binding proteins of 105 kilodaltons (kDa) that were present only in HL-60/AR cells. Competitive gel shift assays indicated that these factors were related to transcription factor Sp1, and immunoblotting with an Sp1 antibody identified this factor as Sp1. DNase footprinting of the promoter region in the human immunodeficiency virus type 1 5' long terminal repeat showed that protection occurred at two Sp1 sites as well as two NF kappa B sites and the trans-acting region with nuclear extracts only from resistant cells. Preliminary evidence also suggests that phosphorylation may play a negative regulatory role in the activity of Sp1, since calf intestine alkaline phosphatase stimulated the DNA-binding activity of Sp1 in vitro. These results indicate that HL-60/AR cells contain an abundance of DNA-binding proteins, particularly Sp1, which probably interact with other cis-acting regulatory proteins in a cooperative manner.

Elevated expression of the c-fes proto-oncogene in adult human myeloid leukemia cells in the absence of gene amplification.

Expression of the 93-kd tyrosine kinase encoded by the human c-fes proto-oncogene (also known as FES) is restricted to mature hematopoietic cells of the granulocytic and monocytic lineages, suggestive of a function essential to normal myeloid differentiation. However, recent studies have shown that c-fes can transform fibroblasts if sufficient levels of gene expression are achieved. These findings indicate that strict regulation of the c-fes gene is critical to normal myeloid development, whereas elevated c-fes expression may contribute to malignant transformation. In the present study, we compared the c-fes messenger RNA (mRNA) levels in leukemia blasts from patients with myeloid or lymphoid leukemia with those of peripheral monocytes from a normal donor with the use of a quantitative ribonuclease protection assay. The presence of c-fes mRNA was readily detected in both acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) cells, but c-fes mRNA was present in low levels or was absent in lymphoid leukemia cells. The leukemia cells of two of five AML patients and four of four CML patients expressed more c-fes mRNA than monocytes from a normal donor, with more than a threefold elevation in the cells of one CML patient. No evidence of amplification or rearrangement of the c-fes gene was detectable by Southern blot analysis of myeloid leukemia DNA, suggesting that the variation in c-fes mRNA levels are related to differences in transcriptional activity and/or message stability. These results indicate that elevated c-fes expression is a common feature of myeloid leukemia cells that could potentially contribute to the leukemia phenotype.

Liposome-mediated modulation of multidrug resistance in human HL-60 leukemia cells.

BACKGROUND: Multidrug resistance (MDR) is a major obstacle in cancer treatment. Resistance of cultured tumor cells to major classes of cytotoxic drugs is frequently due to expression of a plasma membrane P-glycoprotein encoded by MDR genes. We have demonstrated that liposome-encapsulated doxorubicin is more toxic than the free drug and that it modulates MDR in Chinese hamster LZ cells and human colon cancer cells. PURPOSE: To investigate further the association between expression of P-glycoprotein and modulation of MDR by liposome-encapsulated doxorubicin, we studied vincristine-resistant HL-60/VCR leukemia cells, which express P-glycoprotein, and doxorubicin-resistant HL-60/ADR leukemia cells, which do not. METHODS: Cells were exposed to various concentrations of free doxorubicin and liposome-encapsulated doxorubicin. The cellular content of doxorubicin was determined by fluorescence analysis, and cytotoxicity was determined by cell growth inhibition. Photoaffinity-labeling studies of P-glycoprotein binding were performed on HL-60/VCR and HL-60/ADR cells and KB-GSV2 cells transfected with the MDR1 gene (also known as PGY1). RESULTS: The concentrations that caused 50% inhibition of growth (IC50) for free doxorubicin in HL-60, HL-60/ADR, and HL-60/VCR cells were 30 nM, 9 microM, and 0.9 microM, respectively. The values for liposome-encapsulated doxorubicin in parental HL-60 cells and HL-60/ADR cells were 20 nM and 9 microM, respectively, indicating little or no sensitization. In contrast, HL-60/VCR cells were fivefold more sensitive to liposome-encapsulated doxorubicin than to free doxorubicin, and IC50 was reduced to 0.17 microM. In HL-60 cells exposed to liposome-encapsulated doxorubicin, intracellular doxorubicin accumulation was less than that seen with free drug. In contrast, in HL-60/VCR cells, accumulation was twofold to threefold higher than that with free doxorubicin. Liposome-encapsulated doxorubicin completely inhibited the photoaffinity labeling of P-glycoprotein by azidopine in membrane vesicles of HL-60/VCR cells, with a potency comparable to that of azidopine, suggesting that circumvention of MDR by liposomes is related to their specific interaction with P-glycoprotein. The studies with KB-GSV2 cells indicated that blank liposomes can directly inhibit photoaffinity labeling of P-glycoprotein. CONCLUSIONS: These results demonstrate the effectiveness of liposome-encapsulated doxorubicin in overcoming resistance in the multidrug-resistant phenotype of HL-60/VCR cells by direct interaction with P-glycoprotein. Furthermore, they indicate that liposome-encapsulated doxorubicin may be an effective treatment for human cancers.

Cerebrospinal fluid levels of 2'-deoxycoformycin after systemic administration in monkeys.

2'-Deoxycoformycin (DCF) is an inhibitor of the enzyme adenosine deaminase (ADA) and has shown promise as an antileukemia agent. For the assessment of the extent to which systemically administered DCF crosses into the central nervous system (CNS), rhesus monkeys were given iv boluses of DCF. Simultaneous blood and cerebrospinal fluid (CSF) samples were assayed for DCF levels at times ranging from 10 minutes to 6 hours after the drug was given. Average peak CSF drug levels of 5.5 X 10(-8) M and 3 X 10(-7) M were reached 1 1/2 - 2 hours following injections of 0.25 and 1.0 mg DCF/kg, respectively. The ratio of peak CSF to simultaneous plasma levels was 1 to 10. Data obtained from a patient who had acute lymphocytic leukemia and who was given iv DCF were comparable. Drug levels achieved within the CSF following iv administration of 0.25 mg DCF/kg are similar to those previously demonstrated to inhibit ADA. These results may be important both for understanding DCF-related CNS toxicity and for designing combination chemotherapy with DCF.

Alterations in the transcriptional capacity of hepatic DNA-dependent RNA polymerase I and II by N-hydroxy-2-acetylaminofluorene.

Using hepatic RNA polymerases I and II, acetylaminofluorene modification of DNA in vitro results in reduction of RNA elongation but not in an altered frequency of incorporation of [alpha32P]ribonucleotides. In contrast, in vivo modification of RNA polymerases by a single dose of N-2-hydroxy-2-acetylaminofluorene results in an alteration of their capacity for transcribing a normal RNA product but does not affect the product size transcribed from normal or carcinogen-modified templates.

Activity and metabolism of 2-beta-D-ribofuranosylthiazole-4-carboxamide in human lymphoid tumor cells in culture.

The antitumor activity of the C-nucleoside, 2-beta-D-ribofuranosylthiazole-4-carboxamide (TR), was investigated in four human lymphoid tumor cell lines in culture. Exposure of the cell lines CCRF-CEM (T-cell leukemia), HUT-78 (cutaneous T-cell lymphoma), NALM-1 (B-cell leukemia), and MOLT-4 (T-cell leukemia) for 72 hr to TR resulted in 50% inhibitory concentration of 30, 27, 7, and 6 microM, respectively. Maximum inhibition of DNA and RNA synthesis occurred 6 hr after drug treatment. The 50% inhibitory concentration of TR among the four cell lines varied from 5 to 8 microM for RNA synthesis and from 4 to 8 microM for DNA synthesis. Nucleotide analyses in MOLT-4 cells after 6 hr of drug exposure to 10 and 100 microM TR revealed increased inosine 5'-monophosphate concentrations which were 18- to 20-fold greater than control levels and a parallel reduction of 82 and 100% in guanosine 5'-monophosphate concentrations. Growth inhibition of MOLT-4 cells by 6 hr exposure to TR was almost fully reversible by addition of 50 microM guanosine to the medium for 18 hr. Analyses by high-pressure liquid chromatography revealed two metabolites of TR in all four cell lines, namely, thiazolecarboxamide riboside 5'-monophosphate and thiazolecarboxamide adenine dinucleotide, the latter of which is a potent inhibitor of inosine-5-'-monophosphate dehydrogenase. These results suggest that the antitumor effects of TR in human tumor cell lines may relate to guanine nucleotide depletion.

Antagonistic effect of polyinosinic-polycytidylic acid on the cell lethality produced by 5-fluorouracil in human colon carcinoma cells in vitro.

The effect of polyinosinic X polycytidylic acid [poly(I) X poly(C)] on the cell lethality produced by 5-fluorouracil [FUra] and 5-fluorouridine [FUrd] was examined in human colon carcinoma cell line HT-29. Pretreatment of cells with poly(I) X poly(C) as well as during exposure to FUra or FUrd resulted in antagonism of the cell lethality generated by the fluoropyrimidines. Antagonism of FUra cytotoxicity was also produced by the 2'-O-methylated analogue, polyinosinic X poly-2'-O-methylcytidylic acid, but not by the individual single-stranded polynucleotides or by the component mononucleotides, cytidine 5'-phosphate and inosine 5'-phosphate. In contrast, cytidine 5'-phosphate blocked the toxicity of FUrd. The antagonism by poly(I) X poly(C) of FUra and FUrd cytotoxicity was related to the inhibition of their metabolism to fluorouridine triphosphate and their incorporation into RNA and not to inhibition of the synthesis of RNA. Antibodies to leukocyte and fibroblast interferons did not reverse the antagonistic activity of poly(I) X poly(C). These results indicate that poly(I) X poly(C) may be interfering with the transport and/or initial metabolism of FUra and FUrd to fluorouridine monophosphate which is independent of the ability of the double-stranded RNA to induce interferon.

Effects of human immune interferon on cell viability, (2',5')oligoadenylate synthesis, and polyamine-dependent protein phosphorylation in human colon carcinoma cells in vitro.

The growth-inhibitory effect of human immune interferon (IFN-gamma) was investigated in human colon carcinoma cell line HT-29. Three-day treatment of HT-29 cells with IFN-gamma (10 to 200 units/ml) resulted in 30 to 90% growth inhibition and 40 to 99% reduction in colony formation. Measurement of DNA, RNA, and protein synthesis following IFN-gamma treatment showed a dose-dependent reduction in all 3 parameters. The associated changes in (2',5')oligoadenylate [(2',5')oligo(A)] pathway were measured under growth-inhibitory conditions. Upon 1-day exposure to 25 to 200 units/ml of IFN-gamma, (2',5')oligo(A) synthetase activity was induced 10- to 15-fold and remained elevated for 3 days, whereas (2',5')oligo(A) phosphodiesterase activity remained unchanged. There was no detectable increase in intracellular (2',5')oligo(A) levels after IFN-gamma treatment, and ribosomal RNA degradation was not observed. Accompanying 1-day treatment with IFN-gamma (100 units/ml) was an induction of a polyamine-dependent protein kinase, which was double-stranded RNA-independent and phosphorylated endogenous polypeptides with molecular weights of 68,000 and 72,000. A similar exposure of cells to IFN-gamma (25 to 100 units/ml) resulted in 30 to 70% inhibition of ornithine decarboxylase activity; however, no significant alteration in intracellular polyamine levels was observed. These data suggest that IFN-gamma-dependent toxicity is not related to (2',5')oligo(A) activation of a latent endoribonuclease but is accompanied by protein phosphorylation, which is, in part, stimulated by exogenous polyamines.

Effects of fibroblast and recombinant leukocyte interferons and double-stranded RNA on ppp(2'-5')An synthesis and cell proliferation in human colon carcinoma cells in vitro.

The antiproliferative effect of human fibroblast interferon (IFN-beta), human recombinant leukocyte interferon (IFN-alpha A), and polyinosinic . polycytidylic acid [poly(I) . poly(C)] was investigated in human colon carcinoma cell line HT-29. Exposure of HT-29 cells for 1 to 3 days with 100 to 2000 units of either interferon preparation resulted in a 30 to 50% inhibition of growth after 3 days. Similar treatment of cells with 100 micrograms per ml poly(I) . poly(C) resulted in progressive inhibition of growth by 50 to 60% in 2 to 3 days. The inhibitory effects of combination of either IFN-beta or IFN-alpha A with poly(I) . poly(C) were additive with up to 80 to 90% inhibition of cell growth after 3 days of exposure. None of the treatment regimens was markedly cytocidal, and only 25 to 30% reduction in colony formation was noted under optimal treatment conditions following 2 to 3 days of drug exposure. Measurements of DNA, RNA, and protein synthesis following interferon treatment indicated a dose-dependent reduction in all three parameters, particularly when interferon was administered with poly(I) . poly(C). The associated changes in the (2',5')oligoadenylate [(2',5')oligo(A)] pathway produced by IFN-beta and IFL-alpha A were measured under growth-inhibitory conditions. A concentration-dependent induction of (2',5')oligo(A) synthetase was produced by IFN-beta or IFL-alpha A with a concomitant decrease in (2',5')oligo(A) phosphodiesterase. Poly(I) . poly(C) alone induced (2',5')oligo(A) synthetase activity but had no effect on the associated activity of phosphodiesterase. The combination of either IFN-beta or IFL-alpha A and poly(I) . poly(C) further reduced (2',5')oligo(A) phosphodiesterase activity. There was no general dose-response correlation between the induction of (2',5')oligo(A) synthetase and the cytostatic activity of interferon treatment alone or in combination with double-stranded RNA.

Effects of N-acetoxy-2-acetylaminofluorene and N'-nitro-N-nitrosoguanidine on nuclear DNA synthesis in rat liver.

A system for the study of DNA synthesis in isolated nuclei is described for sham and regenerating rat liver. The system has been characterized with respect to nuclear purity, conditions for optimum incorporation of [5-methyl-3H]thymidine triphosphate, time course of incorporation, product analysis by neutral and alkaline sucrose gradients, and the effect of exogenously added DNA. No difference in the basal level of activity was detected between nuclei prepared from normal or regenerating liver when isolated 24 hr after operation. However, exogenous activated DNA preferentially stimulated [5-methyl-3H]thymidine triphosphate incorporation in nuclei from regenerating liver. Activated DNA caused to react with the carcinogen N-acetoxy-2-acetylaminofluorene was a less effective primer-template in this system and decreased in a dose-dependent fashion the incorporation of [5-methyl-3H]thymidine triphosphate to below basal levels in nuclei from both normal and regenerating liver. The carcinogen N-methyl-N'-nitro-N-nitrosoguanidine had no inhibitory effect when assayed in this fashion.

Inhibition of the phosphorylation of non-histone chromosomal proteins of rat liver by cordycepin and cordycepin triphosphate.

The effects of cordycepin (3'-deoxyadenosine) and cordycepin triphosphate on phosphorylation of non-histone chromosomal proteins were assessed in isolated hepatic nuclei in vitro. Cordycepin and cordycepin triphosphate competitively inhibited phosphorylation of urea-soluble nuclear proteins with a Ki of 1.2 X 10(-3) and 8 X 10(-5) M, respectively. Isoelectric focusing of urea-soluble proteins indicated that inhibition occurred predominantly in nuclear proteins with isoelectric points of pH 4 to 7. Quaternary aminoethyl-Sephadex chromatography of extracts of nuclei incubated with cordycepin and cordycepin triphosphate also showed inhibition of phosphorylation of non-histone chromosomal proteins with similar isoelectric points, although greater resolution of proteins with isoelectric points of pH 6 to 7 was achieved. RNA polymerase I and II were not affected by cordycepin and cordycepin triphosphate after quaternary aminoethyl-Sephadex chromatography of nuclear extracts incubated with either agent. However, RNA polymerase I and II in isolated nuclei were competitively inhibited by cordycepin triphosphate but not by cordycepin. These results suggest that cordycepin triphosphate, and perhaps cordycepin too, may affect transcription via interference with the phosphorylation of non-histone chromosomal proteins.

Phospholipid- and Ca2+-dependent protein kinase activity and protein phosphorylation patterns in the differentiation of human promyelocytic leukemia cell line HL-60.

The effects of differentiating agents on the activity and phosphorylation pattern produced by phospholipid- and Ca2+-dependent protein kinase (PL-Ca-PK) were examined in human promyelocytic leukemia cell line HL-60. Dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] increased the appearance of mature myelocytic (DMSO and RA) or monocytic [1,25(OH)2D3] cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the appearance of adherent macrophage-like cells. Coincident with the appearance of differentiated cells induced by DMSO, RA, and 1,25(OH)2D3 was an increase in PL-Ca-PK activity. In contrast, TPA treatment resulted in the rapid disappearance of PL-Ca-PK and the induction of phospholipid- and Ca2+- (PL-Ca-) independent protein kinase activity. The phosphorylation pattern resulting from endogenous PL-Ca-PK in extracts from cells treated with DMSO, RA, or 1,25(OH)2D3 showed a prominent phosphorylated protein of molecular weight 37,000 (pp37) and 38,000 (pp38) which was related to the appearance of the myelocyte/monocyte phenotype. pp37 and pp38 were also present in TPA-treated cells, but their phosphorylation was no longer dependent on the presence of phospholipid and calcium. Cells treated with DMSO and RA also exhibited a PL-Ca-dependent pp21 which was barely evident in 1,25(OH)2D3-treated cells and thus represented a myeloid cell marker. Also present was a prominent PL-Ca-dependent pp19 which remained unchanged following treatment with DMSO, RA, and 1,25(OH)2D3, but which diminished markedly in TPA-treated cells. On the other hand, TPA-treated cells exhibited a characteristic pp130 which was antigenically related to the actin binding protein, vinculin. These results indicate that there are characteristic PL-Ca-dependent phosphorylated proteins indicative of mature myelocytic and monocytic cells, as well as PL-Ca-independent phosphorylated proteins characteristic of the macrophage-like phenotype.

Synergistic effect of human immune interferon and double-stranded RNA against human colon carcinoma cells in vitro.

The cytocidal activity of human immune interferon (IFN-gamma) in combination with the synthetic double-stranded RNA, poly(I).poly(C), was investigated in human colon carcinoma cell line HT-29. Three days of treatment with IFN-gamma (10 to 25 units/ml) resulted in 30 to 40% reduction in colony formation, whereas poly(I).poly(C) (25 to 100 micrograms/ml) reduced cell viability by 10 to 20% of control. The lethal effect of the combination of IFN-gamma and poly(I).poly(C) was synergistic wherein 70 to 90% reduction in colony formation was observed. Measurements of DNA, RNA, and protein synthesis after IFN-gamma and poly(I).poly(C) treatment showed a dose-dependent reduction in all three parameters. Recombinant IFN-gamma in combination with poly(I).poly(C) exhibited a similar effect. Studies evaluating the molecular mechanism of IFN-gamma and poly(I).poly(C) toxicity indicate a lack of involvement of the double-stranded RNA-dependent (2',5')oligoadenylate-RNase L and protein kinase pathways; however, the effect appears to be related to the inhibition of ribosomal RNA transcription in this cell line.

Potentiation of the cytocidal effect of human immune interferon by different synthetic double-stranded RNAs in the refractory human colon carcinoma cell line BE.

A human cell line BE, derived from an undifferentiated carcinoma of the colon, was studied for its response to the cytocidal effects of human immune interferon (IFN-gamma) alone and in combination with various double-standard RNAs (dsRNAs). BE cells were moderately refractory to 3-day treatment with IFN-gamma (10 to 300 units/ml) where only 5 to 30% reduction in colony formation occurred. A similar exposure interval to polyriboinosinic.polyribocytidylic acid [poly(I).poly(C)] (100 micrograms/ml) had no detectable effect on colony formation. In contrast, the lethal effect of the combination of IFN-gamma and poly(I).poly(C) was synergistic and this regimen produced a 40 to 80% reduction in colony formation. The cytocidal effects of the combination of IFN-gamma with varying concentrations of the dsRNAs poly(I).poly(C), polyriboadenylic.polyribouridylic acid [poly(A).poly(U)], polyriboinosinic.polyribocytidylic acid stabilized with poly-L-lysine in carboxymethylcellulose [poly(ICLC)], and mismatched dsRNA [rIn.r(C13,U)n] were also examined. The concentration of the dsRNAs producing a 50% decrease in cell viability in combination with IFN-gamma (100 units/ml) was 6 micrograms/ml for poly(I).poly(C), 1 microgram/ml for poly(A).poly(U), 3 ng/ml for poly(ICLC), and 16 micrograms/ml for rIn.r(C13,U)n. DNA, RNA, and protein synthesis in IFN-gamma and poly(I).poly(C)-treated cells were reduced in a dose-dependent manner. However, there were no changes in either (2',5')oligoadenylate concentrations or in ribosomal RNA transcription following treatment with IFN-gamma and poly(I).poly(C). Thus, the synergism resulting from the combination of IFN-gamma and dsRNA appears to be mediated via another, as yet unknown, mechanism.

Biochemical and morphological changes in hepatic nuclear membranes produced by N-hydroxy-2-acetylaminofluorene.

The effect of N-hydroxy-2-acetylaminofluorene on the ultrastructure and synthesis of hepatic neclear membranes was evaluated in partially hepatectomized rats. The incorporation of L-[4,5-3H]leucine into two nuclear membrane fractions increased within 2 hr after hepatic resection and reached a peak at 20 hr. After partial hepatectomy, the decay of radioactivity in nuclear membrane proteins labeled with L-[4,5-3H]leucine revealed similar half-lives for the two membrane fractions when compared to those obtained from sham-operated animals. The protein concentration of the nuclear membrane fraction of higher density decreased sharply within 2 hr after partial hepatectomy and remained low throughout a 20-hr postoperative period. Polyacrylamide gel electrophoresis of both nuclear membrane fractions showed a similar composition. Nine proteins were resolved, varying from 21,000 to 190,000 daltons. The two major protein bands were in the range of 50,000 and 70,000 daltons, respectively. Treatment of partially hepatectomized animals with N-hydroxy-2-acetylaminofluorene showed marked dilation of the nuclear envelope and rough endoplasmic reticulum in situ upon electron microscopic examination. Vacuolization and evagination of the perinuclear membranes were also noticeable in isolated nuclei obtained from carcinogen-treated rats. Inhibition by N-hydroxy-2-acetylaminofluorene of the incorporation of L-[4,5-3H]leucine into the nuclear membranes was dose-dependent and remained depressed throughout a 60-min labeling period. These results suggest that the inhibitory effects on RNA and protein synthesis previously shown to be produced by this arylhydroxylamine hepatocarcinogen may lead to disruption of the morphology and synthesis of the nuclear envelope.

Synergistic antitumor effect of fluoropyrimidines and polyinosinic-polycytidylic acid against L1210 leukemia.

The effect of polyriboinosinic X polyribocytidylic acid [poly(I) X poly(C)] on the antitumor activity of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) was evaluated in mice bearing L1210 leukemia. Coadministration intravenously of poly(I) X poly(C) and either FUra or FUrd on days 1,5, and 9 to mice bearing L1210 leukemia implanted subcutaneously resulted in a 40% greater increase in life span at the optimal antitumor dose versus FUra and FUrd alone. This effect appeared to result from greater host tolerance of a dose of FUra or FUrd which would otherwise be cytotoxic. The protective effect of poly(I) X poly(C) was also evident in non-tumor-bearing mice, as well as following administration of drug intraperitoneally to mice bearing the tumor implanted intraperitoneally. FUrd incorporation into RNA in the spleen, bone marrow, and small intestine revealed little or no changes after coadministration of poly(I) X poly(C). (2', 5')Oligo(A) synthetase activity, an indication of interferon activity, was markedly depressed in the spleen and bone marrow following treatment with FUrd; however, poly(I) X poly(C) administered together with FUrd returned (2', 5')oligo(A) synthetase activity to normal levels. These data indicate that poly(I) X poly(C) ameliorates the host toxicity of fluoropyrimidines, possibly via an interferon-mediated effect, and thereby results in enhanced therapeutic efficacy of the antimetabolite as an antitumor agent.

Cytokinetic and biochemical effects of 5-iminodaunorubicin in human colon carcinoma in culture.

The semisynthetic anthracycline, 5-iminodaunorubicin (IM), was investigated to see whether modification of the benzoquinone moiety to produce a drug with low free radical potential would alter the cytotoxic and biochemical characteristics of this drug in comparison to Adriamycin (ADR), an agent with high free radical potential. Cell viability was measured in human colon carcinoma (HT-29) cells by soft-agar cloning. Upon exposure to either log-phase or early plateau-phase cells for 2 hr to IM or ADR, a threshold exponential cell lethality curve was obtained. Prolonging during exposure to 24 hr produced an exponential decline in cell survival and a marked reduction in viability of both log-phase and early plateau-phase cells. Inhibition of DNA synthesis in log-phase cells after 2 and 24 hr of exposure to IM and ADR paralleled the increased cell lethality produced by the drugs. In contrast, total RNA synthesis was not inhibited by IM, whereas ADR impaired both RNA and DNA synthesis. Nuclear rRNA, synthesis was not significantly inhibited following 24 hr of exposure to 10(-7) M ADR or IM but was inhibited by 85 and 35% at 10(-6) M ADR or IM, respectively. The affinity of IM and ADR for HT-29 DNA was measured in vitro by displacement of acridine orange binding and was found to be similar for both analogs. These studies suggest that the cytotoxicity of IM and ADR results from the interactions of these drugs with DNA.

Potentiation by 2'-deoxycoformycin of the inhibitory effect by 3'-deoxyadenosine (cordycepin) on nuclear RNA synthesis in L1210 cells in vitro.

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin, on the inhibitory effect of cordycepin on nuclear RNA synthesis was examined in L1210 cells in vitro. The median inhibitory dose for the effect of deoxycoformycin on adenosine deaminase was 4 X 10(-8) M, and 100% inhibition was achieved at 5 X 10(-7) M. Pretreatment of cells for 30 min with 1 X 10(-6) M deoxycoformycin resulted in a reduction of the median inhibitory dose for cordycepin from 2.5 X 10(-4) to 1.8 X 10(-5) M, as assessed by the measurement of [3H]uridine incorporation into total RNA. Measurement of the synthesis of nuclear ribosomal RNA, nonpolyadenylic acid heterogeneous RNA, and polyadenylic acid heterogeneous RNA revealed potentiation by 2'-deoxycoformycin of the inhibitory effect of cordycepin on all species of RNA, as well as on polyadenylic acid synthesis. No differences were noted in the size of nuclear polyadenylic acid obtained from cells treated with cordycepin in either the presence or the absence of the adenosine deaminase inhibitor. These results suggest that the potentiation by 2'-deoxycoformycin of the cytotoxic and antitumor effects of cordycepin on L1210 cells in vivo is related to inhibition of nuclear RNA synthesis.

Akt1 induces extracellular matrix invasion and matrix metalloproteinase-2 activity in mouse mammary epithelial cells.

The roles of the protein-serine/threonine kinase, Akt1, in signaling pathways associated with cell motility and extracellular matrix invasion were examined in the immortalized mouse mammary epithelial cell line, COMMA-1D. COMMA-1D cells were engineered to express the avian leukosis subtype A receptor, tv-a, to permit infection by recombinant avian leukosis virus produced by the replication-competent avian splice vector, RCAS. COMMA-1D/tv-a cells transduced with RCAS/v-akt, but not RCAS/Akt1, formed anchorage-independent colonies in soft agar; however, cells overexpressing either v-akt or Akt1 became highly invasive when grown on the ECM, Matrigel. Zymography of extracellular protease activity shed into the medium by COMMA-1D/Akt1 or COMMA-1D/v-akt cells revealed elevated gelatinase activity that was confirmed to be matrix metalloproteinase-2 (MMP-2; gelatinase A) by Western blotting and immunoprecipitation-zymography. The MMP inhibitor, BB-94, blocked MMP-2 activity and invasion associated with Akt1- and v-akt-expressing cells. The proteasome inhibitor, lactacystin, markedly increased MMP-2 levels and invasion in control cells but not in Akt1- and v-akt-expressing cells. These results suggest that the invasive behavior of mammary epithelial cells induced by Akt1 is associated with increased MMP-2 expression that may result from inhibition of MMP-2 degradation by the proteasome pathway.

Kinetic effects of sangivamycin in sarcoma 180 in vitro.

The lethal and sublethal effects of sangivamycin (SGM) were studied in sarcoma 180 in vitro in relation to drug concentration and duration of drug exposure. SGM lethality was found to be dependent on both drug concentration and duration of drug exposure. Pronounced effects on cell survival were observed only when SGM exposure was prolonged; with prolonged drug exposure, small increments in SGM concentration resulted in large increases in cell killing. Log-phase cells were more susceptible to the lethal effects of SGM than were early-plateau-phase cells. Measurements of incorporation of [3H]thymidine and [3H]uridine into the acid-insoluble cell fraction demonstrated inhibition of both DNA and RNA synthesis by SGM which was also dependent on drug concentration and duration of drug exposure, reflecting the lethality characteristics of SGM. As SGM concentration was increased, DNA synthesis was inhibited more rapidly than was RNA synthesis. Flow cytometry demonstrated a concentration- and time-dependent accumulation of cells in the late S and G2-M region of the DNA histogram. Our findings indicate that maximum lethality is obtained by prolongation of SGM exposure, and they suggest that pharmacokinetic studies may be important for determining regimens which provide such exposure in humans.