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3.
J Immunol Methods ; 243(1-2): 99-106, 2000 Sep 21.
Article in English | MEDLINE | ID: mdl-10986409

ABSTRACT

An increased level of complexity will be encountered when developing protocols for intracellular markers. Protocols for surface markers have been successfully standardized, however it is understood that no single method is appropriate for all intracellular staining. A systematic approach should be followed, including knowledge of antigen location and functional state, selection of cell fixative and cell permeabilizer, antibody specificity and class/subclass, fluorochrome, fluorochrome to protein ratio (F:P), and use of adequate controls, including isotype-matched negative controls and positive and negative cell controls. Even though it is impossible to recommend a single technique to stain all intracellular antigens, the authors present a logical approach to follow when developing a staining protocol.


Subject(s)
Biomarkers/analysis , Antibodies/immunology , Antigens, Surface/analysis , Cell Membrane Permeability , Cross-Linking Reagents/chemistry , Detergents/chemistry , Fixatives , Flow Cytometry/methods , Fluorescent Dyes , Humans
4.
Clin Chem Lab Med ; 37(3): 311-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10353477

ABSTRACT

Three documented cell death pathways, apoptosis, necrosis, and oncosis will be discussed. The end result of each pathway is cell death; however, the path by which death is achieved and the morphological and physiological traits of each may be strikingly distinct. Now that well characterized models have been established for particularly apoptosis, the induction pathway(s) has received much attention and the pathway pathology is beginning to be understood. Three model systems were investigated: APO-1/Fas, hypoxia, and oncosis. Cell death was induced, and during a time course sampling, a variety of methodologies, including DNA fragmentation by flow cytometry and gel electrophoresis, DNA staining, flow cytometric light scatter, transmission electron microscopy, anti-tubulin, Trypan blue, annexin V, and anti-APO2.7 were employed to monitor the cell death progress. The apoptotic pathway in the CD95-induced Jurkat cell model was further investigated using caspase inhibitor peptides and analyzed for APO2.7 antigen expression and DNA fragmentation by flow cytometry. Time course sampling characterized the cell death pathway and helped to differentiate the capabilities of the methods. The time to response and duration of the response were dependent upon cell type and method of induction. The CD95-induced Jurkat cell model showed a classical apoptotic response; however the MDA-MB-175-VII hypoxia model and the anti-5A9 induced oncosis model were not as clear. Each methodology shows advantages and disadvantages that allow the investigator to select several methods to identify, monitor, and enumerate cells with respect to cell death progression using time course studies.


Subject(s)
Cell Death , Cell Differentiation , Cell Death/immunology , Flow Cytometry , Humans , Light , Membrane Lipids/metabolism , Microscopy, Electron , Phospholipids/metabolism , Scattering, Radiation , Signal Transduction , Tumor Cells, Cultured
5.
Cytometry ; 33(3): 324-32, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9822343

ABSTRACT

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.


Subject(s)
Antibodies, Monoclonal , Cell Death/physiology , Cell Hypoxia/physiology , Membrane Proteins/analysis , Mitochondria/immunology , Apoptosis/physiology , Cell Survival/physiology , Flow Cytometry/methods , Humans , Intracellular Membranes , Necrosis , Time Factors , Tumor Cells, Cultured
6.
Mol Cell Biol ; 18(1): 85-92, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9418856

ABSTRACT

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.


Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Carcinoma/genetics , Enzyme Inhibitors/pharmacology , Mammary Neoplasms, Experimental/genetics , Methionine/analogs & derivatives , Salivary Gland Neoplasms/genetics , Animals , Antineoplastic Agents/therapeutic use , Carcinoma/drug therapy , Carcinoma/pathology , Cell Cycle/drug effects , Enzyme Inhibitors/therapeutic use , Farnesyltranstransferase , Female , Genes, ras , Humans , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/pathology , Mammary Tumor Virus, Mouse , Methionine/pharmacology , Methionine/therapeutic use , Mice , Mice, Transgenic , Salivary Gland Neoplasms/drug therapy , Salivary Gland Neoplasms/pathology
7.
Cytometry ; 28(1): 81-9, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9136759

ABSTRACT

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.


Subject(s)
Apoptosis , Breast Neoplasms/pathology , DNA, Neoplasm/isolation & purification , Mammary Neoplasms, Experimental/pathology , Tubulin/metabolism , Animals , Breast Neoplasms/metabolism , Cell Hypoxia , Cell Separation , Cell Survival , Electrophoresis, Agar Gel , Flow Cytometry , Fluorescent Antibody Technique , Humans , Mammary Neoplasms, Experimental/metabolism , Mice , Necrosis , Ploidies , Propidium , Tubulin/immunology , Tumor Cells, Cultured
8.
Cancer Res ; 57(4): 600-3, 1997 Feb 15.
Article in English | MEDLINE | ID: mdl-9044833

ABSTRACT

We have used the MMTV-myc and MMTV-ras transgenic mouse mammary tumor models (T. A. Stewart et al., Cell, 38: 627-637, 1984, and E. Sinn et al., Cell, 49: 465-475, 1987) to evaluate how the c-myc and v-Ha-ras oncogenes influence tumor growth characteristics in vivo. MMTV-myc tumors had much higher levels of spontaneous apoptosis than MMTV-ras tumors, whereas intermediate levels were observed in MMTV-myc/ras tumors. Significant differences in cell cycle characteristics were also observed in tumors from mice of the three genotypes. Tumors from MMTV-myc mice had lower G1 and higher S-phase fractions than MMTV-ras tumors, with intermediate values again observed in the MMTV-myc/ras tumors. Despite these differences, however, tumor growth rates for the different groups were similar. These findings highlight the importance of the balance between cell cycle regulation and cell death in determining the kinetics of tumor growth and indicate that distinct oncogenes can have a profound influence on that balance.


Subject(s)
Apoptosis/genetics , Genes, cdc/physiology , Genes, myc/physiology , Genes, ras/physiology , Mammary Neoplasms, Experimental/genetics , Mammary Tumor Virus, Mouse/genetics , Animals , Cell Cycle/genetics , Cell Division , Genotype , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic
9.
Mol Cell Biol ; 17(2): 723-31, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9001226

ABSTRACT

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.


Subject(s)
Genes, ras/physiology , Mammary Neoplasms, Experimental/genetics , Mammary Tumor Virus, Mouse , Salivary Gland Neoplasms/genetics , Tumor Suppressor Protein p53/physiology , Tumor Virus Infections/genetics , Aneuploidy , Animals , Apoptosis , Cell Division , Crosses, Genetic , Female , Genetic Heterogeneity , Genotype , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Mice, Transgenic , Salivary Gland Neoplasms/pathology , Tumor Suppressor Protein p53/genetics , Tumor Virus Infections/pathology
10.
Biochem Pharmacol ; 54(11): 1181-93, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9416969

ABSTRACT

6-Hydroxymethylacylfulvene (HMAF, MGI 114) is a new alkylating antitumor sesquiterpenoid with promising and often curative antitumor activity in vivo. This study examined the ability of the drug to damage cellular DNA, induce apoptosis, and affect the cell cycle of CEM human leukemia cells. No bifunctional lesions, interstrand DNA cross-links or DNA-protein cross-links were seen (by alkaline sedimentation and K+/SDS precipitation, respectively) when using up to 50 microM HMAF. The drug possibly formed some monoadducts, as DNA from drug-treated cells impeded primer extension by Taq polymerase, although only partial inhibition was seen even at 200 microM HMAF. HMAF also induced secondary lesions in cellular DNA, single-strand breaks that were detectable (by nucleoid sedimentation and alkaline sucrose gradient analysis) after a 4-hr treatment at HMAF levels as low as 2 microM, comparable to the growth inhibition IC50 value (1.7 microM). A post-treatment incubation of cells in drug-free medium generated substantial amounts of DNA double-stranded fragments of several kbp, suggesting apoptotic fragmentation (>30% of total DNA following treatment with 20 microM HMAF and a 17-hr post-treatment incubation). Chromatin condensation (by ultrastructural analysis) and induction of sub-G1 particles and apoptotic strand breakage (by multiparametric flow cytometry) confirmed induction of apoptosis by HMAF. HMAF preferentially inhibited DNA synthesis (IC50 approximately 2 microM), which is consistent with an S phase block, observed by cell cycle analysis. The pattern of apoptotic DNA fragmentation, inhibition of DNA synthesis, and blockage in the S phase suggests that these events play a role in the antiproliferative activity of HMAF.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , DNA/drug effects , Sesquiterpenes/pharmacology , Cell Cycle/drug effects , DNA/biosynthesis , DNA Damage , DNA Fragmentation , Flow Cytometry , Humans
11.
Cytometry ; 29(4): 306-12, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9415413

ABSTRACT

A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.


Subject(s)
Antibodies, Monoclonal , Apoptosis , Jurkat Cells/pathology , Membrane Proteins/immunology , Mitochondria/immunology , Annexin A5/metabolism , Apoptosis/drug effects , Biomarkers , DNA Fragmentation , DNA, Neoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Flow Cytometry/methods , Humans , Jurkat Cells/metabolism , fas Receptor/pharmacology
12.
Breast Cancer Res Treat ; 32(1): 57-65, 1994.
Article in English | MEDLINE | ID: mdl-7819587

ABSTRACT

Flow cytometric analysis of DNA ploidy and S-phase fraction are well recognized prognostic indicators in breast cancer. The present paper deals with the widening of the applications of flow cytometry to monitoring the effectiveness of antiestrogen therapy, detecting clonal selection and emergence of drug resistance, and monitoring chemosensitizing properties of drugs. Antiestrogen activity can be studied by DNA flow cytometry to address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance. Patient plasma specimens containing various concentrations of triphenylethylenes can be monitored for drug-induced effects using cell cycle measurements and correlated to in vivo drug levels. DNA flow cytometry has also been instrumental in the study of the effects of prolonged low-dose (0.5 microM for > 100 days) tamoxifen treatment on human estrogen receptor negative MDA-MB-231 cells, where it was shown that tamoxifen may significantly alter cell cycle kinetics and tumorigenicity of these cells, selecting a new, more aggressive, and rapidly growing clone. Lastly, it has been shown that the chemosensitizing properties of another triphenylethylene antiestrogen, toremifene, on estrogen receptor negative, multidrug resistant MDA-MB-231-A1 human breast cancer cells can be studied using flow cytometric analysis. Toremifene (and its metabolites N-desmethyltoremifene and toremifene IV) are able to "resensitize" MDA-MB-231-A1 cells to vinblastine and doxorubicin, as reflected in a marked shift of cells to G2/M phase of the cell cycle. Flow cytometry is a widely available technique that might be applied clinically to monitor, at the cellular level, drug effects on tumors, including the modulators of drug resistance.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , DNA, Neoplasm/analysis , Flow Cytometry , Antineoplastic Agents/therapeutic use , Cell Cycle/drug effects , Drug Resistance, Multiple , Estrogen Antagonists/therapeutic use , Humans
14.
Oncol Res ; 5(12): 461-6, 1993.
Article in English | MEDLINE | ID: mdl-8086667

ABSTRACT

The in vivo growth rate and the chemosensitivity patterns of a cell clone selected by tamoxifen from the estrogen receptor-negative human breast cancer cell line MDA-MB-231 was studied in the nude mouse model and with flow cytometry. To investigate the growth rate of the wild-type and clone cells in vivo, the cells were inoculated into the opposite flanks of 5 male nude mice. Drug sensitivity to doxorubicin (10 ng/mL), vinblastine (1 ng/mL), and paclitaxel (1 ng/mL) was examined in wild-type/clone cell mixture using flow cytometry. Northern blot technique was used to study the expression of mdr-1 messenger RNA in both the wild-type and the clone cells. The tumors derived from the clone and wild-type cells were, following a 3-week growth period, 260.2 +/- 78.8 mm2 vs. 68.3 +/- 50.8 mm2 in size, respectively (P < 0.001). Following a 28-day continuous exposure, doxorubicin was selectively, toxic to the wild-type cells, while having no apparent effect on the clone population. However, paclitaxel- and vinblastine-treated wild-type/clone cell mixtures did not exhibit a differential cytotoxic effect on either cell population. It was concluded that the clone selected by tamoxifen shows an aggressive growth rate in vivo and an altered chemosensitivity pattern to doxorubicin in vitro.


Subject(s)
Breast Neoplasms/pathology , Doxorubicin/pharmacology , Tamoxifen/pharmacology , Animals , Cell Division/drug effects , Drug Resistance , Female , Humans , Male , Mice , Mice, Inbred BALB C , Tumor Cells, Cultured
15.
Eur J Cancer ; 29A(15): 2138-44, 1993.
Article in English | MEDLINE | ID: mdl-8297653

ABSTRACT

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.


Subject(s)
Breast Neoplasms/pathology , Neoplastic Stem Cells/drug effects , Tamoxifen/pharmacology , Animals , Breast Neoplasms/chemistry , Cell Division/drug effects , Female , Flow Cytometry , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Proteins/analysis , Neoplasm Transplantation , Receptors, Estrogen/analysis , Time Factors , Transplantation, Heterologous , Tumor Cells, Cultured
16.
Oncol Res ; 5(6-7): 207-12, 1993.
Article in English | MEDLINE | ID: mdl-8123940

ABSTRACT

The clinical study of compounds that modulate multidrug resistance has been hindered by both the toxicities of these agents and the inability to monitor their effectiveness at the level of the tumor cell. Previously, toremifene has been shown to be well tolerated clinically and to sensitize multidrug resistant cells to the effects of cytotoxic chemotherapeutic agents. The chemosensitizing properties of toremifene in estrogen receptor negative, multidrug resistant MDA-MB-A1 human breast cancer cells were studied using flow cytometric analysis and growth inhibition assays. Cell cycle kinetics of MDA-MB-A1 cells were not significantly affected by treatment with either toremifene, N-desmethyltoremifene, Toremifene IV or vinblastine alone, as the majority of cells remained in G0/G1. However, preincubation with toremifene or one of its metabolites for 72 hours followed by treatment for one hour with vinblastine caused a marked shift of cells to G2/M, as cells appeared to be blocked in that phase of the cell cycle. This result was nearly identical to the effect of vinblastine alone on vinblastine-sensitive MDA-MB-231 breast cancer cells and can be interpreted as a "resensitization" by toremifene of MDA-MB-A1 cells to vinblastine. This chemosensitizing effect of toremifene was accompanied by an enhanced inhibition of cell growth by vinblastine. The chemosensitizing effects of toremifene or one of its metabolites in combination with cytotoxic chemotherapy can be effectively monitored by flow cytometry, an easily accessible technique.


Subject(s)
Breast Neoplasms/pathology , Drug Resistance , Toremifene/pharmacology , Vinblastine/pharmacology , Breast Neoplasms/drug therapy , Cell Cycle/drug effects , Cell Division/drug effects , Drug Synergism , Female , Flow Cytometry , Humans , Tumor Cells, Cultured
17.
Breast Cancer Res Treat ; 24(1): 43-9, 1992.
Article in English | MEDLINE | ID: mdl-1463871

ABSTRACT

The clinical study of compounds that modulate multidrug resistance in cancer cells has been hindered by both the toxicities of these agents and the inability to monitor their effectiveness at a cellular level. The non-steroidal triphenylethylene toremifene is well tolerated clinically and can sensitize multidrug resistant cells to the effects of doxorubicin in vitro. The chemosensitizing properties of toremifene in estrogen receptor negative, multidrug resistant MDA-A1 human breast cancer cells were studied using flow cytometric analysis. Cell cycle kinetics of MDA-A1 cells were not significantly affected by treatment with either toremifene or doxorubicin alone, as the majority of cells remained in G0/G1. However, preincubation with toremifene for 70 hours followed by treatment with doxorubicin caused a marked shift of cells to G2, as cells appeared to be blocked in that phase of the cell cycle. This result was nearly identical to the effect of doxorubicin alone on doxorubicin-sensitive MDA-MB-231 breast cancer cells and can be interpreted as a "resensitization" by toremifene of MDA-A1 cells to doxorubicin. This chemosensitizing effect of toremifene was accompanied by an enhanced accumulation of doxorubicin in MDA-A1 cells (+110% after 70 hours pre-incubation with toremifene), and by a depression in protein kinase C activity in MDA-A1 cells that was maximal following 70 hours incubation with toremifene. Flow cytometry is a widely available technique that might be applied clinically to monitor at the cellular level the chemosensitizing effects of toremifene and other modulators of multidrug resistance.


Subject(s)
Breast Neoplasms/pathology , Receptors, Estrogen/analysis , Toremifene/pharmacology , Breast Neoplasms/metabolism , Cell Cycle/drug effects , Doxorubicin/pharmacokinetics , Drug Resistance , Flow Cytometry , Humans , Protein Kinase C/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism
18.
Invest New Drugs ; 8 Suppl 1: S9-18, 1990.
Article in English | MEDLINE | ID: mdl-1696247

ABSTRACT

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with alpha-difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.


Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/pharmacology , Eflornithine/pharmacology , Stomach Neoplasms/drug therapy , Adenocarcinoma/pathology , Antineoplastic Agents/administration & dosage , Bleomycin/administration & dosage , Bleomycin/therapeutic use , Cell Cycle/drug effects , Cell Survival/drug effects , Doxorubicin/administration & dosage , Doxorubicin/therapeutic use , Drug Administration Schedule , Drug Therapy, Combination , Eflornithine/administration & dosage , Fluorouracil/administration & dosage , Fluorouracil/therapeutic use , Humans , Ornithine Decarboxylase/metabolism , Polyamines/metabolism , Semustine/administration & dosage , Semustine/therapeutic use , Stomach Neoplasms/pathology , Time Factors , Tumor Cells, Cultured/drug effects
19.
Invest New Drugs ; 6(4): 293-8, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3229942

ABSTRACT

We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3-4) to MeCCNU (10 micrograms/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 micrograms/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 micrograms MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably.


Subject(s)
Astrocytoma/pathology , Carmustine/pharmacology , Cell Survival/drug effects , Clone Cells/drug effects , Clone Cells/radiation effects , Doxorubicin/pharmacology , Drug Resistance , Galactitol/pharmacology , Humans , Semustine/pharmacology
20.
Cancer Res ; 48(10): 2749-55, 1988 May 15.
Article in English | MEDLINE | ID: mdl-3359435

ABSTRACT

An in vitro model has been devised so that mixtures of human tumor cells can be grown together for studies related to drug-induced or -selected changes in sensitivity. In the studies reported here, two human astrocytoma clones, one sensitive and one resistant to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU), were carefully matched for doubling times, cell cycle phase distributions, and colony-forming efficiencies. The clones were mixed and grown together, and after only three weekly treatments with MeCCNU (10 micrograms/ml for 1 h each week) the sensitive cells in the mixture were killed, leaving behind a population that was almost 100% resistant to further exposures to MeCCNU. The loss of the sensitive cells from the mixture each week was easily detected by visual observation of flow microfluorometry histograms since the clones had different DNA indices. Repeated weekly exposures of the unmixed resistant clone (AST 1-1) to MeCCNU caused very little accumulated cell kill. Similar exposures of the unmixed sensitive clone (AST 3-4) produced a linear decrease in survival over the first three weekly treatments with 10 micrograms MeCCNU/ml, but after that time these cells became progressively more resistant to MeCCNU. It is unlikely that the change to resistance in the AST 3-4 clone occurred because of contamination with the resistant AST 1-1 cells, because their DNA index remained stable. These data show that repeated treatments with a single agent can cause a tumor cell population to become more resistant. It remains to be tested whether this resistance was the result of cellular interactions, drug-induced changes in sensitivity, or selection for resistant cells already present in the populations. This mixture model may be useful in studies on how cellular interactions influence growth and drug sensitivity in tumor and normal cell populations.


Subject(s)
Tumor Cells, Cultured/drug effects , Astrocytoma/pathology , Cell Survival/drug effects , DNA, Neoplasm/analysis , Dose-Response Relationship, Drug , Drug Resistance , Humans , Models, Biological , Semustine/pharmacology
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