Methotrexate cytotoxicity in cultured human leukemic cells studied by flow cytometry.

Methotrexate (MTX)(2 x 10(-8) M) inhibited DNA synthesis in CCRF-CEM cells, causing cells to accumulate in early S phase while cellular RNA content and cell size continued to increase. Two-parameter flow cytometric analysis of DNA and RNA showed these cells to be unbalanced with excessive RNA relative to DNA content. Fifty % of cells remained viable after a 96-hr exposure to 2 x 10(-8) M MTX. In contrast, 10(-4) M MTX inhibited cell cycle progression of cells in both G1 and S phases and also prevented the development of unbalanced growth. In this instance, cell viability was reduced to 10% after 96 hr of drug exposure. The relative contribution of inhibition of thymidylate and purine biosynthesis to MTX cytotoxicity was investigated by addition of exogenous thymidine (10(-5) M) or hypoxanthine (10(-4) M). Thymidine reduced the cytotoxicity and inhibition of DNA synthesis caused by both doses of MTX and prevented classical unbalanced growth with 2 x 10(-8) m MTX; treatment with 10(-4) M MTX resulted in a form of unbalanced growth where cells had a relative excess of DNA compared with RNA content. The addition of hypoxanthine enhanced the classical unbalanced growth pattern seen with 2 x 10(-8) M MTX but was accompanied by a partial reduction of both the MTX-induced cytotoxicity and the inhibition of DNA synthesis (to an extent similar to that seen with exogenous thymidine). Potentiation of cell killing was observed with the addition of hypoxanthine to cells treated with 10(-4) M MTX. Complete rescue from MTX cytotoxicity at both concentrations was found only when both thymidine and hypoxanthine were present. These finding suggest that MTX cytotoxicity is associated with inhibition of DNA synthesis resulting from the disturbance of both thymidylate and purine biosynthesis.

Flow cytometric analysis of adenosine analogue lymphocytotoxicity.

The induction of G1-phase arrest in T-lymphoblasts by cytostatic concentrations of 2'-deoxyadenosine (R. M. Fox, R. F. Kefford, E. H. Tripp, and I. W. Taylor, Cancer Res., 41: 5141-5150, 1981) prompted a flow cytometric analysis of the cell cycle effects of three other adenosine analogues with known effects on polyadenylated RNA metabolism in an attempt to further explore the nature of 2'-deoxyadenosine 5'-triphosphate-mediated lymphotoxicity. Cytostatic concentrations of 9-beta-D-arabinofuranosyladenine induced an S-phase block, while 3'-deoxyadenosine (cordycepin) and tubercidin (7-deazaadenosine) induced a cycle-nonspecific block. Furthermore, total cellular RNA content was unaltered by 2'-deoxyadenosine or 9-beta-D-arabinofuranosyladenine, but 3'-deoxyadenosine and tubercidin caused a marked reduction in total cellular RNA at minimally cytostatic concentrations. At concentrations of 0.3 to 20 microM, all of these nucleosides were toxic to nondividing peripheral blood lymphocytes, suggesting that in these cells their mechanism of action does not involve reactions associated with DNA replication. Inhibition of polyadenylated RNA metabolism by triphosphate derivatives of adenosine analogues may account for lymphocytotoxicity in nondividing cells, but the demonstrated diverse effects of these nucleosides on nucleic acid metabolism in dividing cells preclude elucidation of the mechanism of the unique induction of G1-phase arrest by 2'-deoxyadenosine.

Cell proliferation kinetics of MCF-7 human mammary carcinoma cells in culture and effects of tamoxifen on exponentially growing and plateau-phase cells.

MCF-7 human mammary carcinoma cells were inoculated into 150-sq cm flasks at 3 X 10(5) cells/flask, and after a lag period of about 48 hr, these cells grew exponentially for 5 days with a mean population doubling time of about 24 hr. During exponential growth, 80 to 90% of cells were in the "rapidly cycling" pool, the clonogenic fraction was 50 to 60%, and the mean percentage of cells in the G0-G1, S, and G2 + M phases of the cell cycle was 48.9 +/- 0.6% (S.E.), 39.4 +/- 0.6%, and 11.6 +/- 0.3%, respectively. These parameters changed rapidly between Days 7 and 13 when plateau phase was reached. Between Days 13 and 18, 74.8 +/- 0.7% of cells were in G0-G1, 15.3 +/- 0.4% were in S, and 9.8 +/- 0.6% were in G2 + M phase. Only about 30% of these cells were cycling rapidly, and the clonogenic fraction had fallen to less than 10%. Tamoxifen induced a dose-dependent decrease in the growth rate of exponentially growing cells, which was accompanied by a dose-dependent increase in percentage of G0-G1-phase cells, and a decline in percentage of S-phase cells. At doses greater than or equal to 10 microM, a 24-hr pulse of tamoxifen was cytotoxic to exponentially growing cells. Plateau-phase cells were less sensitive to these effects of tamoxifen. In an attempt to define the kinetic basis of the G0-G1 accumulation induced by tamoxifen, asynchronous MCF-7 cells were pretreated for 42 hr with various doses of tamoxifen, and the rate of efflux of cells from the G0-G1 phase of the cell cycle was assessed by blocking their reentry into G1 with ICRF 159. Following treatment of control cultures with ICRF 159, two populations of cells were distinguished by their rates of efflux from G0-G1 phase. The majority of cells left G0-G1 rapidly with a mean t1/2 of 2.3 hr ("rapidly cycling" cells). However, about 18% of cells had a much slower rate of exit with a mean t1/2 of about 30 hr ("slowly cycling" cells). Pretreatment with tamoxifen resulted in a dose-dependent decrease in the proportion of rapidly cycling cells and an increase in the proportion of cells with slow G1 transit times. Although this appeared to be the predominant effect, tamoxifen also decreased the rate at which the slowly cycling cells traversed G1. Simultaneous treatment with estradiol returned these parameters to control values at doses of tamoxifen less than or equal to 5 microM, partially reversed the effect of 7.5 microM tamoxifen, but was without effect on the arrest of cell cycle progression induced by 10 microM tamoxifen. It is concluded that cells accumulate in G0-G1 following tamoxifen treatment due to an increase in the proportion of slowly cycling cells at the expense of a population of rapidly cycling cells, which appear to be relatively uninfluenced by the drug.

Effects of tamoxifen on cell cycle progression of synchronous MCF-7 human mammary carcinoma cells.

The effects of tamoxifen on cell cycle progression and clonogenic survival have been examined using synchronized cultures of MCF-7 human mammary carcinoma cells. Cell synchrony was induced by mitotic selection. Subsequent cell cycle analyses, using DNA flow cytometry, showed that 85% of synchronized cells had a mean cell cycle time of 21.3 hr with mean phase durations of 9 hr for G0-G1, 9.3 hr for S, and 3 hr for G2 + M. A slowly cycling or noncycling subpopulation comprising 15% of the total population was also observed. Exposure to tamoxifen (5 to 12.5 microM) resulted in a dose-dependent reduction in the number of cells progressing through G0-G1 and entering S phase. Those cells which were not retained in G0-G1, however, appeared to traverse G0-G1 and the remainder of the cell cycle at a rate only slightly less than that of untreated controls. Further experiments demonstrated that the major sensitivity to tamoxifen in terms of both inhibition of cell cycle progression and drug cytotoxicity was restricted to a short interval in the middle of G0-G1. This 2- to 4-hr period of maximum drug sensitivity began approximately 4 hr after mitotic selection, with drug exposures outside this time frame having markedly fewer effects. The significance of these observations in the light of previous studies with asynchronous populations of MCF-7 cells is discussed.

Purine modulation of methotrexate cytotoxicity in mammalian cell lines.

The modulation of MTX cytotoxicity by purines has been studied in a number of mammalian cell lines. In each case, it was found that exogenous purines (guanosine, deoxyguanosine, adenosine, deoxyadenosine, and hypoxanthine) both reduced and potentiated MTX cytotoxicity depending on the MTX concentration. At low MTX concentrations (less than 6 X 10(-8) M), purines reduced MTX toxicity and at higher concentrations they potentiated MTX toxicity. The reduction of low-concentration MTX cytotoxicity by purines was associated with the reversal of MTX-induced changes in deoxyribonucleotide pools. On the other hand, potentiation of MTX toxicity by purines was associated with substantial increases in deoxyadenosine 5'-triphosphate levels in conjunction with low deoxythymidine 5'-triphosphate levels. The magnitude of increase in deoxyadenosine 5'-triphosphate levels tended to correlate with the degree of potentiation which varied between 5-fold and 200-fold, depending on cell type and the exogenous purine.

Influence of cellular DNA content on disease-free survival of Stage II breast cancer patients.

Using a novel flow cytometric method to analyze paraffin-embedded archival material, cellular DNA content of the primary tumor was measured in 165 patients with Stage II breast cancer who had been entered onto a large, multicenter trial of adjuvant chemotherapy. Fifty-three (32%) of the tumors examined were diploid, and the remainder contained one or more aneuploid clones. Aneuploid tumors had more extensive axillary lymph node involvement (p less than 0.05 using chi 2 analysis), but there were no significant correlations between cellular DNA content and either menopausal or estrogen receptor status. Forty-nine of 112 (44%) patients with aneuploid tumors have relapsed, compared to 12 of 53 (23%) with diploid tumors, and relapse-free survival curves show that beyond 2 years the diploid group reaches an apparent plateau, with a projected 4-year relapse-free survival of 72%, whereas the aneuploid group shows a continuing risk of relapse with only 43% remaining relapse free at 4 years. This correlation was most pronounced in the premenopausal patients; only 5 of 36 (14%) of those with diploid tumors have relapsed compared to 23 of 63 (37%) in the aneuploid group. However, multivariate analysis using a stepwise Cox model does not thus far confirm an independent prognostic significance of cellular DNA content on disease-free survival compared to node status. The cellular DNA content of the primary tumor did not appear to influence the patients' survival following relapse. These results indicate that compared to aneuploid tumors either diploid tumors have a different natural history or they are more responsive to adjuvant chemotherapy, possibly due to a lower probability of their developing drug resistance.

G1-phase arrest of cultured human leukemic T-cells induced by deoxyadenosine.

Cultured human T-cell leukemia lymphocytes have enhanced sensitivity to growth inhibition by deoxyadenosine. We have used flow cytometry to investigate the mechanism of deoxyadenosine toxicity in cultured T-leukemic cells. Comparative studies on deoxyadenosine-resistant Epstein-Barr virus-transformed B-lymphocyte cell lines were also performed. After exposure of T-cells to low concentrations of deoxyadenosine (3 microM), in the presence of an adenosine deaminase inhibitor (erythro-9-[3-(2-hydroxynonyl)]adenosine), accumulation of cells of cells with a G1 DNA content was demonstrated. In contrast, B-cell lines showed a similar degree of growth inhibition after exposure to 200 to 400 microM deoxyadenosine but were blocked in S phase. The T-cell G1 block was associated with a rise in the deoxyadenosine triphosphate pool, and both these phenomena were prevented by the addition of deoxycytidine. The biochemical mechanism of this G1 block induced by deoxyadenosine in T-cells is not understood.

Selective toxicity of a new lipophilic antifolate, BW301U, for methotrexate-resistant cells with reduced drug uptake.

Three methotrexate (MTX)-resistant cell lines and their MTX-sensitive counterparts have been used to examine 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methyl-pyrido[2,3-d]pyrimidine (BW301U), a novel lipophilic antifolate, and compare its cytotoxicity with MTX and metoprine. Collateral sensitivity for both BW301U and metoprine was observed in CCRF-CEM/MTX R-cells, where MTX resistance appeared to be primarily due to a deficiency in drug uptake. This was particularly pronounced with BW301U which proved to be as effective in killing CCRF-CEM/MTX R as was MTX with the parental CCRF-CEM cell line. This effect was not seen in other cell lines, L5178Y/MTX or L1210/MTX R, where resistance to MTX was correlated with either an overproduction of 5,6,7,8-tetrahydrofolate:nicotinamide adenine dinucleotide phosphate oxidoreductase EC 1.5.1.3 (DHFR) or with combined uptake defect and increased DHFR levels, respectively. In each case, however, BW301U and metoprine, especially at high concentrations, were more effective than MTX in treating MTX-resistant cells.

Influence of cellular DNA content on survival in advanced ovarian cancer.

Tumor cellular DNA content was measured by flow cytometry in 91 patients with advanced ovarian cancer, using a new and simple technique which allows archival paraffin-embedded tissue to be studied; 69% of the tumors were aneuploid, and 31% were diploid. Tumor ploidy was shown by multivariate Cox model analysis to be an independent prognostic variable and the major determinant of survival. Patients with diploid tumors survived significantly longer than did those with aneuploid tumors (p much less than 0.0001; X2 = 25.44; d.f. = 1).

A rapid single step staining technique for DNA analysis by flow microfluorimetry.

A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.

An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations.

Several preparative techniques (detergent treatment, ethanol fixation, and hypotonic cell lysis), DNA fluorochromes, and methods of numerical analysis (planimetric or curve-fitting) were compared for the estimation of cell-cycle kinetic parameters (G1, S, G2 + M) by flow cytometry. In addition, coefficients of variation (CV), relative fluorescence, and G1/chicken erythrocyte (CRBC) ratios were measured and the effects of the proportion of cycling cells and cellular RNA content were examined. DNA fluorochromes were ranked by relative fluorescence: 4,6-diamidino-2-phenylindole > ethidium bromide/mithramycin > Hoechst 33342 > mithramycin > ethidium bromide > acridine orange approximately equal to propidium iodide. The first four (DNA-specific stains) gave lower CVs than the remainder (DNA intercalators). Detergent treatment also increased relative fluorescence and slightly lowered CVs. Comparable results were obtained for the kinetic parameters independently of stain or staining procedure; intercalating dyes with cells of a high RNA content not treated with RNAse and acridine orange being the exceptions. Of the two methods of numerical analysis, the planimetric technique was more consistant. Although highly consistant G1/CRBC ratios were obtained for any one stain, independently of staining procedures, variations between stains were noted. It is suggested that the detergent treatment in combination with DNA-specific stains provide optimal results.

Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.

A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.

Elimination of glyceryl trinitrate: effects of sex, age, species and route of administration.

1 Orally administered glyceryl trinitrate to rats undergoes extensive first pass metabolism leading to low bioavailability. 2 Sex differences in the plasma elimination of glyceryl trinitrate were seen in the rat, the female exhibiting the longer plasma half-life. No sex differences in this respect were detected in the rabbit. 3 The plasma half-life of glyceryl trinitrate was longer and the volume of distribution larger, in older animals. 4 The plasma elimination of glyceryl trinitrate was different in various animal species. There was a good correlation between plasma half-life and animal bodyweight.

Effect of deuteration of imipramine on its pharmacokinetic properties in the rat.

Imipramine was specifically deuterated in either both aromatic rings or in the N-methyl group, or in both positions, and the pharmacokinetic properties of the products were determined in the rat and compared with those of the non-deuterated analogue. Deuteration of imipramine resulted in a small but significant isotope effect on N-demethylation while aromatic hydroxylation was unaffected. This isotope effect led to a slower rate of systemic clearance, a longer half-life and, when orally administered, enhanced bioavailability. Urinary excretion of didesmethylimipramine-d4, following oral administration of imipramine-d7, was significantly lower than the excretion of didesmethylimipramine following administration of unlabelled imipramine, indicating inhibited demethylation. Similarly, the urinary excretion of desmethylimipramine-d4, didesmethylimipramine-d4 and 2-hydroxydesmethylimipramine-d4 were lower than for the corresponding unlabelled or d7-analogues, indicating the stability of the N-CD3 group. Deuteration had no effect on the pharmacological properties of imipramine as determined in this study.

Clinical and biological significance of aneuploidy in human tumours.

Aneuploidy is a well recognised feature of human tumours, but the investigation of its biological and clinical significance has been hampered by technological constraints. Quantitative DNA analysis reflects the total chromosomal content of tumour cells and can now be determined rapidly and reliably using flow cytometry; this has resulted in renewed interest in its potential clinical applications. This article reviews the accumulating evidence that tumour ploidy reflects the biological behaviour of a large number of tumour types and that diploid tumours in particular have a relatively good prognosis. The measurement of tumour ploidy is likely to become a valuable adjunct to the clinical and histopathological assessment of cancers.

Flow cytometric analysis of cellular DNA content as an adjunct to the diagnosis of ovarian tumours of borderline malignancy.

Flow cytometric analysis of cellular DNA content was performed on tissue from 44 borderline ovarian tumours (tumours of low malignant potential). Forty-two tumours (95%) were diploid and associated with both an indolent biological behaviour and good prognosis. Aneuploidy was identified in only 2 tumours (5%), and one of the associated patients died of progressive disease within months of the initial diagnosis. Careful review of the histopathology of these 2 aneuploid tumours revealed areas of invasion in the omental and peritoneal "implants" of each. This study has reinforced the currently advocated separation of so-called borderline tumours from invasive ovarian carcinomas and the interpretation of the pathological criteria used to categorize such neoplasms. Our results indicate that flow cytometric analysis of cellular DNA content may complement conventional histopathological diagnosis by providing an objective parameter which correlates with biological behaviour and may identify the few genuine borderline ovarian epithelial neoplasms which show clinical progression.