Patterns of pancytopenia in Yemen.

OBJECTIVE: The aim of this study was to determine the incidence of pancytopenia in relation to sex and age and to become aware of the clinical manifestations of pancytopenic patients according to the causes of pancytopenia. MATERIALS-METHODS: This was a prospective descriptive study that included all patients with pancytopenia admitted to or attending the Hematology-Oncology Department in Al-Gamhouria Teaching Hospital, Aden, during the period 1 January-31 December 2005. RESULTS: The most common causes of pancytopenia were malaria and hypersplenism in > 45% of patients, followed by megaloblastic anemia in 14.7%, and aplastic anemia and acute leukemia in 13.3% each. The other causes as determined in the present study were myelodysplasia in 8.0%, myelofibrosis in 4.0% and iron deficiency anemia in 1.3%. The most common symptom was fever in 86.7% of patients. Pallor and splenomegaly were the most common physical findings. CONCLUSIONS: Hypersplenism and malaria were the most common causes of pancytopenia followed by megaloblastic anemia, aplastic anemia and acute leukemia.

Doxorubicin-induced ultrastructural and growth changes in primary cultures of 7,12-dimethylbenz[a]anthracene-induced mammary tumors.

For the determination of their response to doxorubicin (Dx) (adriamycin), monodispersed mammary tumor cells (from female outbred Sprague-Dawley rats) were maintained as monolayer primary culture. Dose-response curves and [3H]thymidine labeling indices showed the antimitotic and cytocidal effects of the drug varied in a dose-dependent fashion. Dose-response curves revealed that the median lethal dose concentration was 10(-4) M. A 24-hour treatment at concentrations of 10(-4) to 10(-5) M completely arrested DNA synthesis of the tumor cells. Surviving cells exhibited chromatin abnormalities, accumulation of cytoplasmic myelin bodies, vacuolization of endoplasmic reticulum, and increased density of mitochondrial matrix. This study showed 1) 7,12-dimethylbenz[a]anthracene-induced mammary tumor cells were highly sensitive to Dx; 2) Dx induced specific ultrastructural effects on the nuclei, mitochondria, and membranes of the cells; and 3) the in vitro response of the primary culture may be useful for prediction of the response of the source tumor to chemotherapy.

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.

Light, fluorescent, and electron microscopic analysis of cultured breast tumor cells (T-47D) treated with 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazol-2-yl)hydrazone] dihydrochloride.

The influence of bisantrene on T-47D human breast tumor cells was assessed by colony-forming assay in soft agar and by light, fluorescence, and electron microscopy. Test solutions of bisantrene solubilized in distilled water or dimethyl sulfoxide were added to cultures at final concentrations between 0.01 and 60 micrograms/ml. Brightly fluorescent particles appeared in a concentration-dependent fashion after cultures were treated with water-soluble bisantrene at concentrations greater than 0.1 microgram/ml. Similar fluorescent crystals appeared in culture media when concentrations of the dimethyl sulfoxide-dissolved drug exceeded 10 micrograms/ml. Clonogenic survival as defined by soft agar assay indicated significant reproductive impairment in cells treated with concentrations greater than 1 micrograms/ml (p less than 0.01). Nuclear and cytoplasmic fluorescence was evident in treated cells. Cells that survived 24-hr drug treatment displayed round nuclei with watery nucleoplasm when examined under the light microscope. Under the electron microscope, nuclei of these cells revealed fragmentation of the nucleolar complex and a highly electron-lucent nucleoplasm. Cytoplasmic responses, which seem to be relatively innocuous, include incorporation of the fluorescent crystals into lysosomes and some mitochondrial abnormalities. Crystalline inclusions engulfed by lysosomes were found in cells obtained from cultures showing bisantrene precipitates.

Identification of Vinca alkaloid acceptors in P388 murine leukemia cells with a photoactive analogue of vinblastine.

The cytotoxic, antimitotic, and growth inhibition properties of a photoactive analogue of vinblastine, N-(p-azidobenzoyl)-N'-beta-aminoethylvindesine (NABV), and vinblastine on P388 murine leukemia cells were compared. After 72-h exposure, the 50% drug-inhibitory concentrations for exponentially growing P388 leukemic cells were 1.2 nM for NABV and 0.6 nM for vinblastine. The ultrastructural effects of NABV and vinblastine on P388 cells were similar: formation of tubulin paracrystals; mitotic arrest (C-mitosis); increased post-C-mitotic multinucleated cells; increased number of annulate lamellae; and the appearance of intracytoplasmic paired cisternae. [3H]NABV was used to identify Vinca alkaloid binding sites in P388 cells by photoaffinity labeling. After irradiation at 302 nm, radioactive Vinca alkaloid binding components were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified in 1-mm gel slices. The most prominent photolabeled species were Mr 44,000, 54,000, and 75,000 polypeptides located in the 100,000 X g supernatant fraction. The Mr 54,000 component was also observed in the membrane fraction. Specific photolabeling of Mr 54,000 and 44,000 polypeptides was blocked in the presence of 20 microM excess of vinblastine and was saturable with half-maximal saturation concentrations of 0.18 and 0.4 microM [3H]NABV, respectively. The Mr 54,000 component was identified as a tubulin subunit by immunoprecipitation with antitubulin monoclonal antibodies. Since NABV and vinblastine have similar pharmacological and biological properties, this photoactive analogue may be useful for identifying important Vinca alkaloid cellular acceptors which may be responsible for drug cytotoxic and antineoplastic activities.

Pharmacokinetic modulation of irinotecan and metabolites by cyclosporin A.

The focus of this investigation was to modulate the pharmacokinetics of irinotecan and its metabolites, SN-38 and SN-38G, by possibly reducing biliary excretion, which in turn could lower irinotecan toxicity. We determined the effect of a known cholestatic agent, cyclosporin A (CsA), which is transported across the bile canalicular membrane by P-glycoprotein, on the biliary excretion of irinotecan and its metabolites. Wistar rats were pretreated with 60 mg/kg CsA 5 min before an i.v. dose of irinotecan at dose levels of 6, 10, and 20 mg/kg. The control groups received irinotecan only. CsA pretreatment resulted in an average increase of 339, 361, and 192% in the area under the plasma concentration-time curve of irinotecan, SN-38, and SN-38G, respectively. Analysis of clearance (CL) of irinotecan indicated a 55 and 81% reduction in the average renal and nonrenal CLs, respectively, in the pretreated groups. The nonrenal CL, which is the primary component of irinotecan CL, includes protein and tissue binding as well as the metabolic and biliary CL of irinotecan. There was no change in the volume of distribution at steady state (indicative of unchanged binding) and in the metabolic conversion of irinotecan to SN-38 due to pretreatment. Therefore, the significant reduction in the systemic CL of irinotecan due to CsA pretreatment was primarily due to lowered biliary excretion. Studies using a photoaffinity analogue of verapamil, [125I]NAS-VP, and membrane vesicles from the multidrug-resistant cell line, MCF-7/Adr, revealed that irinotecan and metabolites had moderate interaction with P-glycoprotein. Further studies are required to determine the mechanism of inhibitory effect of CsA on the biliary excretion of irinotecan and its metabolites.

Characterization of monoclonal antibodies recognizing a Mr 180,000 P-glycoprotein: differential expression of the Mr 180,000 and Mr 170,000 P-glycoproteins in multidrug-resistant human tumor cells.

P-glycoprotein is a plasma membrane protein believed to mediate resistance to natural product drugs such as vincristine, Adriamycin, and actinomycin D. To facilitate the study of human P-glycoprotein, monoclonal antibodies (designated HYB-612, HYB-241, and HYB-195) were raised against vincristine-resistant human neuroblastoma (SH-SY5Y/VCR) cells. The antibodies recognize a Mr 180,000 plasma membrane phosphoglycoprotein produced in increased amounts in SH-SY5Y/VCR as well as in vincristine-resistant human neuroepithelioma (MC-IXC/VCR), vinblastine-resistant human leukemia (CEM/VLB100), and actinomycin D- or vincristine-resistant Chinese hamster (DC-3F/AD X and DC-3F/VCRd-5L) cells, as compared to control cells. Radioimmunoprecipitation of proteins in cells metabolically labeled with [35S]methionine, 32Pi, or [3H]glucosamine and Western transfer procedures were used for these studies. Characterization of the HYB-612 or HYB-241 antigen by destructive degradation produced a pattern of results typical of a conformation-dependent protein epitope. HYB-612 recognizes complexes of the Mr 180,000 antigen with an iodinated photoaffinity analogue of vinblastine or with tritiated azidopine. Furthermore, pretreatment of MC-IXC and MC-IXC/VCR cells with HYB-612 or HYB-241 before measurement of tritium-labeled actinomycin D or vincristine uptake increases the amount of drug accumulation in resistant, but not in sensitive, cells. Of importance is the fact that the Mr 180,000 protein is expressed in cells which also contain a Mr 170,000 P-glycoprotein. The relative amounts of the Mr 180,000 and 170,000 species vary from one drug-resistant cell line to another. Evidence that the Mr 180,000 protein is a P-glycoprotein and that there is a conserved complex pattern of resistance-related surface proteins in multidrug-resistant cells is presented in this report.

Pharmacological, molecular, and cytogenetic analysis of "atypical" multidrug-resistant human leukemic cells.

We previously described the cross-resistance patterns and cellular pharmacology of a human leukemic cell line, CEM/VM-1, selected for resistance to the epipodophyllotoxin teniposide (M. K. Danks et al., Cancer Res., 47: 1297-1301, 1987). Compared to CEM/VLB100, which is a well characterized "classic" multidrug-resistant (MDR) cell line, the CEM/VM-1 cells display "atypical" multidrug resistance (at-MDR) in that they are cross-resistant to a wide variety of natural product antitumor drugs, except the Vinca alkaloids, and they are not impaired in their ability to accumulate radiolabeled epipodophyllotoxin. We have extended our characterization of this at-MDR cell line in the present study. In comparison to CEM/VLB100 cells, we found that CEM/VM-1 cells are not cross-resistant to either actinomycin D or colchicine. Verapamil and chloroquine, which enhance the cytotoxicity of vinblastine in CEM/VLB100 cells, had little or no ability to do so in the CEM/VM-1 cells. Membrane vesicles of the two resistant sublines were examined for overexpression of the MDR-associated plasma membrane protein (P-glycoprotein, Mr 170,000 protein, or 180,000 glycoprotein) by photoaffinity labeling with the vinblastine analogue N-(p-azido[3-125I]salicyl)-N'-beta-aminoethylvindesine. We were unable to visualize the MDR-associated protein in the CEM/VM-1 membranes with this photoaffinity probe under conditions in which the P-glycoprotein was readily seen in the membranes of CEM/VLB100 cells. Furthermore, no hybridization of the pMDR1 complementary DNA was seen in slot-blot analyses of the RNA from at-MDR cells, indicating that the mdr gene coding for P-glycoprotein is not overexpressed as is the case in the classic MDR cells. However, cytogenetic analysis indicated that the CEM/VM-1 cells contained an abnormally banded region on chromosome 13q, suggesting that a gene other than mdr may be amplified in these cells. Thus, despite the two cell lines having approximately equal degrees of resistance to epipodophyllotoxins, our data indicate that the mechanism(s) responsible for at-MDR is different from that for classic, P-glycoprotein-associated MDR.

Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance.

Several mechanisms of drug resistance have been defined using cell lines selected for resistance in vitro. However, the relevance of these to tumor cell resistance in vivo remains unclear. We established tumor cell lines from biopsies of human sarcomas before and after doxorubicin therapy. One pretreatment sarcoma line, STSAR90, was 6-fold less sensitive to doxorubicin than was a normal fibroblast line, AG1522. The sensitivities of six other sarcoma lines were similar to that of AG1522. STSAR90 cells did not overexpress P-glycoprotein mRNA, by Northern analysis with the pCHP1 complementary DNA fragment. Photoaffinity labeling with the vinblastine analogue N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine did not show increased P-glycoprotein concentrations. Accumulation of [3H]daunomycin was not decreased in STSAR90 compared with a less resistant sarcoma line, STSAR11, nor was the doxorubicin sensitivity of STSAR90 increased by coincubation with verapamil. Glutathione levels were twice as high in STSAR90 as in STSAR11, and glutathione peroxidase activity was 3.5- to 6-fold higher. This was due mostly to an increase in selenium-dependent peroxidase activity. After exposure to doxorubicin, STSAR90 cells formed only half as much measurable hydroxyl radical as STSAR11, as detected by electron spin resonance spectrometry. Doxorubicin sensitivity was increased in STSAR90 cells when intracellular glutathione levels were reduced by buthionine sulfoximine. These results indicate that multidrug resistance due to P-glycoprotein-mediated drug efflux is not the only mechanism of doxorubicin resistance that occurs in sarcomas and that glutathione peroxidase-dependent detoxification of doxorubicin-induced oxygen radicals may contribute to clinical doxorubicin resistance.

Expression of the mutated p53 tumor suppressor protein and its molecular and biochemical characterization in multidrug resistant MCF-7/Adr human breast cancer cells.

Multidrug resistance in MCF-7/Adr human breast cancer cells is mediated by several mechanisms including overexpression of the MDR1 gene product, P-glycoprotein and glutathione-related detoxifying enzymes. Mutations in the p53 tumor suppressor protein have been reported to play a role in the development of resistance to DNA damaging agents in several human cancer cells. In the present study we have assessed the mutational status of the p53 protein and its expression levels, degree of stability and cellular localization to investigate whether it is involved in modulating multidrug resistance in MCF-7/Adr cells compared to sensitive MCF-7 cells. As revealed by immunofluorescence microscopy using the anti-p53 mouse monoclonal antibody DO-1, wild-type p53 is sequestered in the cytoplasm of MCF-7 cells, whereas in MCF-7/Adr cells, the protein is localized in the nucleus. The sequencing of full-length p53 cDNA revealed a 21 bp deletion in its one of the four conserved regions within the conformational domain, spanning codons 126-133 at exon five, in MCF-7/Adr cells. Moreover, detection of ThaI polymorphism of codon 72 showed that MCF-7 cells predominantly express wild-type p53 with proline, while mutated p53 in MCF-7/Adr cells contains an arginine residue at codon 72. In addition, we demonstrate that the half-life of p53 in MCF-7 cells is less than 30 min while the mutated protein is more stable; its half-life is about 4 h in MCF-7/Adr cells. Thus, this study demonstrates that the deletion of codons 126-133 in p53 causes increased stability, overexpression and nuclear localization of the protein in multidrug resistant MCF-7/Adr cells, and further suggests that mutated p53 might be involved in the development of multidrug resistance in this cell line.

beta(2)-microglobulin induces apoptosis in HL-60 human leukemia cell line and its multidrug resistant variants overexpressing MRP1 but lacking Bax or overexpressing P-glycoprotein.

In this study, we examined whether exogenous beta(2)-microglobulin (beta(2)m) can induce apoptosis in the drug sensitive HL-60 leukemia cell line and its drug resistant variants and investigated the molecular mechanism of beta(2)m-induced apoptosis. Our data revealed that beta(2)m is very significantly down-regulated in two multidrug resistant variants of the HL-60 cells: (a) the MRP1-bearing, Bax-deficient HL-60/ADR cell line, and (b) the P-glycoprotein (P-gp) overexpressing HL-60/VCR cell line. However, exogenous beta(2)m induced similar levels of apoptosis in HL-60 cells and these drug resistant variants. beta(2)m-induced apoptosis in HL-60 and HL-60/VCR cells was associated with decreased mitochondrial membrane potential (Deltapsim) but did not affect Deltapsim in HL-60/ADR cells. Surprisingly, cyclosporin A (CsA), a known inhibitor of the mitochondrial permeability transition (MPT) pore, inhibited beta(2)m-induced apoptosis in HL-60/ADR cells but not in HL-60 and HL-60/VCR cells, suggesting that the pro-apoptotic effect of beta(2)m in these cells is not through MPT pore formation. Furthermore, beta(2)m induced the release of cytochrome c and the apoptosis-inducing factor (AIF) from mitochondria in HL-60 and HL-60/VCR cells, but not in HL-60/ADR cells. Additionally, Z-VAD-fmk, a general inhibitor of caspases which inhibited cytochrome c release in HL-60 and HL-60/VCR cells, had no effect on AIF release in any of these cell lines, but inhibited beta(2)m-induced apoptosis in all three cell lines. However, Western blot analysis revealed that caspases-1, -3, -6, -8, and -9 are not activated during beta(2)m-induced apoptosis in these cells. Therefore, beta(2)m-induces apoptosis through an unknown caspase-dependent mitochondrial pathway in HL-60 and HL-60/VCR cells and by a Bax-independent, non-mitochondrial, caspase-dependent pathway in HL-60/ADR cells.

Modulation of vinblastine resistance with cyclosporine: a phase I study.

OBJECTIVE: Tumor cell resistance is a major cause of failure to cure advanced malignancies. Multidrug resistance is thought to be an important mechanism of such resistance. Our aims were to identify doses of cyclosporine that would achieve blood levels effective in modulating multidrug resistance to vinblastine and to evaluate the toxicities and maximum tolerated dose of cyclosporine when administered in conjunction with vinblastine. METHODS: We conducted a phase I trial of vinblastine and escalating doses of cyclosporine. Cyclosporine was given by continuous intravenous infusion over 120 hours and vinblastine was administered by continuous infusion from hour 12 to hour 108. Sixty-two patients entered the trial, of whom 60 were evaluable. RESULTS: Cyclosporine was escalated from 1 to 15.6 mg/kg/day. Vinblastine doses were reduced to 1.6 and then 1.2 mg/m2/day because of increasing vinblastine toxicity at higher cyclosporine doses. The maximum tolerated dose of cyclosporine at 1.2 mg/m2/day vinblastine was 12.5 mg/kg/day; at this dose level, mean blood cyclosporine level was 1.25 +/- 0.41 mumol/L. Significant nephrotoxicity was observed at higher cyclosporine doses in two of four patients. Nephrotoxicity was not significant at doses at or lower than this maximum tolerated dose and was not cyclosporine dose dependent. Myelosuppression, neurotoxicity, and transient hyperbilirubinemia were observed and were cyclosporine dose dependent. CONCLUSIONS. Cyclosporine by continuous infusion may be safely given in high doses concurrently with continuous-infusion vinblastine. Plasma levels of cyclosporine > or = 1 mumol/L can be sustained during vinblastine administration. No sustained effect on T-cell subsets was observed. Vinblastine toxicity is enhanced by cyclosporine in a dose-dependent fashion and correlates with cyclosporine-induced hyperbilirubinemia.

Single-agent gemcitabine and gemcitabine/irinotecan combination (irimogem) in non-small cell lung cancer.

Gemcitabine is a fluoridated pyrimidine related to cytosine arabinoside that has significant activity in solid tumor models. Irinotecan is a camptothecin analog with an active metabolite, SN-38, which inhibits topoisomerase I activity by stabilizing the topoisomerase I-DNA cleavable complex. Gemcitabine studies in non-small cell lung cancer conducted in the United States, as well as an international collaboration and clinical trials from Europe and Japan, found overall response rates of 20% to 26%, a median duration of response between 5 to 9 months, and a median duration of survival ranging from 7 to 12.3 months. Gemcitabine also has been shown to be more effective than best supportive care in non-small cell lung cancer. In a phase I trial of irinotecan (50, 75, 100, and 115 mg/m2) in combination with 1,000 mg/m2 gemcitabine, three patients had documented partial responses: one with pancreas cancer at irinotecan 100 mg/m2, one with pancreas cancer, and one with metastatic carcinoma of unknown primary at irinotecan 115 mg/m2. Three of five non-small cell lung cancer patients had stable disease for four or more cycles at irinotecan doses of 50, 75, and 100 mg/m2; no non-small cell lung cancer patients were treated at irinotecan 115 mg/m2. We recommend that a combination of gemcitabine 1,000 mg/m2 and irinotecan 100 mg/m2 given on days 1 and 8 every 3 weeks be used as the starting dose in future phase II studies. Furthermore, based on the absence of severe nonhematologic toxicity or grade IV hematologic toxicity in the majority of patients treated at the highest dose, escalation of irinotecan to 115 mg/m2 may be considered for subsequent cycles in patients who do not experience > or =grade I hematologic or non-hematologic toxicity during the first cycle of gemcitabine/irinotecan combination chemotherapy.

Inhibition of protein synthesis and cell proliferation in cultured human breast cancer cells treated with mitoxantrone.

Mitoxantrone suppresses cell proliferation, inhibits protein synthesis and induces ultrastructural alterations in the T-47D and MDA-MB-231 breast cancer cell lines. After 24 h treatment with 10(-9), 10(-7) and 10(-5) M drug and 8 h incubation with [35S]methionine, protein synthesis declined rapidly. While a 10-15% decrease in protein synthesis at 10(-9) M was observed, more than 95% inhibition of protein synthesis occurred at 10(-5) M mitoxantrone in both cell lines. Sodium dodecylsulfate (SDS) gel electrophoresis of labeled proteins revealed no qualitative changes in either cell line. However, only trace amounts of several proteins were present in T-47D cells treated with 10(-5) M drug. At 10(-9) M mitoxantrone had little effect on cell proliferation. At 10(-7) M, 25% and 35% growth inhibition in T-47D and MDA-MB-231 cells was observed, respectively. Cell growth at 10(-5) M was abolished. Cytotoxicity was evident at drug concentrations above 10(-5) M. Ultrastructural alterations in the nucleoli of both cell lines included disintegration and segregation of granular and fibrillar components and the disappearance of nucleolar organizers at 10(-7)-10(-5) M mitoxantrone.

Influence of mitoxantrone on nucleolar function in MDA-MB-231 human breast tumor cell line.

The effect of mitoxantrone (DHAQ) on [3H]thymidine and [3H]uridine incorporation by exponentially growing MDA-MB-231, a human breast tumor cell line has been studied. The results have indicated that DHAQ was more effective in inhibiting [3H]thymidine than [3H]uridine incorporation in a concentration dependent manner. Following drug treatment at 20 ng/ml concentration, 50% inhibition of growth and [3H]thymidine incorporation were noted, whereas [3H]uridine incorporation was only inhibited by about 12%. At 2000 ng/ml of DHAQ the inhibition of cell growth, [3H]thymidine and [3H]uridine incorporations were 78%, 95% and 62%, respectively. Nuclear-associated radioactivity detected at light and electron microscope autoradiographic levels after [3H]thymidine and [3H]uridine incorporations, into DHAQ treated cells indicated that DHAQ prevented the accumulation of radioactivity into the nuclei in a concentration dependent manner. These results gave further indication that mitoxantrone induced a definitive alteration of nuclear template activities, correlated with nuclear functional-structural relation and suggested that the nucleoli were the primary site of DHAQ action.

Morphological alterations induced by prostaglandins E1, F2 alpha and A1 in MDA-MB-231 and MCF-7 human breast cancer cell lines.

Monolayer cultures of MDA-MB-231 and MCF-7 human breast tumor cell lines were treated with prostaglandins PGE1, PGF2 alpha and PGA1 in a concentration range of 10(-12)-10(-4) M and studied at ultrastructural level. Electron microscopic examinations of both cell lines revealed that PGE1, PGF2 alpha and PGA1 induced morphological changes at concentrations above 10(-8) M. In both the small and large MDA-MB-231 cells, deformation of mitochondrial cristae, increased density of mitochondrial matrix and accumulation of lysosomal-like vesicles were observed. In the nuclei morphological, modifications included, the presence of nuclear bodies, occasional nuclear inclusions, nucleolar budding and the disappearance of the nucleolar granular components. In MCF-7 cells, disorganization of mitochondrial cristae and an increase in their matrix density were also observed. At nuclear level, little or no morphological alterations were observed. The results also indicated that the plasma membranes of both cell lines were the most sensitive organelles to PGs action as in many cells their microvilli were either shortened and spherical in shape or absent.

Acute effects of mitoxantrone on the template activity of isolated nuclei from the T-47D human breast tumor cell line.

The effect of mitoxantrone on the template activity of nuclei isolated from the T-47D human breast tumor cell line was investigated. The results suggest that mitoxantrone significantly inhibits total RNA synthesis of these nuclei in a concentration-dependent manner. At low drug concentrations (10(-9) M and 10(-7) M) RNA synthesis was inhibited by 21.9% and 41% compared to control values, respectively. Greater inhibition was observed when the mitoxantrone concentration was increased to 10(-5) M or 10(-4) M (56% and 77%, respectively). Experiments utilizing alpha-amanitin revealed that mitoxantrone inhibits RNA polymerase II activity in a concentration-dependent manner.

Oestrogen dependence of lactate dehydrogenase and peroxidase in cell subpopulations of 7,12-dimethylbenz[alpha]anthracene-induced mammary tumours in the rat.

Recent investigations of certain enzymes as markers for predicting the response of breast tumours to hormonal therapy have neglected the possible differential contribution of cell subpopulation(s) within a solid tumour to enzyme activity. In this investigation, lactate dehydrogenase (LDH) and peroxidase activities in density-defined cell subpopulations from autotransplanted 7,12-dimethylbenz[alpha]anthracene-induced mammary tumours were determined. The effects of ovariectomy and subsequent oestrogen administration on the activity of these enzymes were also examined. Five cell subpopulations (cell bands) were routinely obtained from each mammary tumour. The highest LDH activity was found in cell band 4. The highest level of peroxidase activity was found in cell band 5. These two cell bands with high levels of enzyme activity consisted mainly of poorly differentiated cells. After bilateral ovariectomy, a significant (P less than 0.001) decrease in the level of LDH activity in cell bands 3, 4 and 5 was observed. The enzyme activity was reduced to 20, 2.1 and 12% of the preovariectomy levels respectively. Significant (P less than 0.05) decreases between baseline and postovariectomy peroxidase values were evident in each cell band. In the presence of oestradiol-17 beta, significant increases in the LDH activity of band 4 (P less than 0.001) and the peroxidase activity of band 5 (P less than 0.05) were observed. Our data suggest that, given the existence of multiple cell types in hormone-responsive tumour tissue, the actual cell subpopulation(s) responsible for any enzyme response may be a more precise indicator of hormone dependence.

Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression.

HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.

Effects of sphingosine stereoisomers on P-glycoprotein phosphorylation and vinblastine accumulation in multidrug-resistant MCF-7 cells.

To investigate the role of protein kinase C (PKC) in the regulation of multidrug resistance and P-glycoprotein (P-gp) phosphorylation, the natural isomer of sphingosine (SPH), D-erythro sphingosine (De SPH), and its three unnatural stereoisomers were synthesized. The SPH isomers showed similar potencies as inhibitors of in vitro PKC activity and phorbol binding, with IC50 values of approximately 50 microM in both assays. Treatment of multidrug-resistant MCF-7ADR cells with SPH stereoisomers increased vinblastine (VLB) accumulation up to 6-fold at 50 microM but did not alter VLB accumulation in drug-sensitive MCF-7 wild-type (WT) cells or accumulation of 5-fluorouracil in either cell line. Phorbol dibutyrate treatment of MCF-7ADR cells increased phosphorylation of P-gp, and this increase was inhibited by prior treatment with SPH stereoisomers. Treatment of MCF-7ADR cells with SPH stereoisomers decreased basal phosphorylation of the P-gp, suggesting inhibition of PKC-mediated phosphorylation of P-gp. Most drugs that are known to reverse multidrug resistance, including several PKC inhibitors, have been shown to directly interact with P-gp and inhibit drug binding. SPH stereoisomers did not inhibit specific binding of [3H] VLB to MCF-7ADR cell membranes or [3H]azidopine photoaffinity labeling of P-gp or alter P-gp ATPase activity. These results suggest that SPH isomers are not substrates of P-gp and suggest that modulation of VLB accumulation by SPH stereoisomers is associated with inhibition of PKC-mediated phosphorylation of P-gp.