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Biomedical subjects

Z Darzynkiewicz

Publications and source records attributed to Z Darzynkiewicz.

At least 289 records · Page 16Linked to original sources
PubMed1981

Rapid analysis of drug effects on the cell cycle.

Using a flow cytometric technique to analyse DNA content and chromatin structure simultaneously, the following parameters of cell cycle progression were estimated in control and drug-treated L1210 cell cultures: (a) the kinetics of cell exit from the G1 phase; (b) the probability of cell exit from the indeterminate portion of the G1 phase, measured as the half-time of cell residence in that state; (c) the duration of the deterministic portion of G1 phase; (d) the rates of cell transit through selected "windows" in S phase; (e) the rate of cell entrance into mitosis; (f) the mean duration of the cell cycle (Tc). These parameters are obtained in a single stathmokinetic experiment from measurements of individual samples withdrawn at 30 min-1 hr intervals from Vinblasatine-treated cultures. In the same experiment mitotic indices are obtained with high statistical accuracy, and may be used to determine the terminal point of drug action. In addition to cell cycle analysis the method makes it possible to detect drug-induced changes in nuclear chromatin that are manifested by varying sensitivity of DNA in situ to denaturation by acid. Such changes were found to be associated with defective chromatin condensation, altered histone modifications or intercalation of the drugs into DNA. Using this technique the effects of sodium n-butyrate and two new antitumor drugs on L1210 cells were investigated.

Animals↗
PubMed1981

Flow cytometry for followup examinations of conservatively treated low stage bladder tumors.

A series of 26 conservatively treated patients with low stage bladder tumors was examined later by outpatient cystoscopy, conventional cytology and flow cytometry. Based on those findings the patients were separated into 5 groups. In the 4 patients in group 1 all findings persistently were negative. The 4 patients in group 2 had initially normal cystoscopy with progressively more abnormal flow cytometry and intermittently positive cytology; they subsequently suffered tumors. The 10 patients in group 3 had positive cystoscopy and flow cytometry, and intermittently positive cytology. The 6 patients in group 4 had negative cystoscopy with intermittently positive cytology and persistently positive flow cytometry. In all 6 cases flow cytometry was more sensitive than conventional cytology in detecting early and, sometimes, cystoscopically occult neoplasms. In the 2 patients in group 5 cystoscopically visible papillomas and low grade papillary carcinomas were found with negative flow cytometry and negative or suspicious cytology. The papillary tumors in these 2 patients were believed to be composed principally of benign or atypical epithelium, with too few malignant cells to be detected by present flow cytometry techniques. Our preliminary results suggest that flow cytometry may be a valuable tool for followup of patients who have had conservatively treated low stage bladder tumors and are at risk for development of carcinoma. In addition, the techniques may be used to monitor changes in the proportion of benign and malignant cells and, thus, can be of help in following the course of newly forming papillomas, papillary carcinomas or carcinoma in situ in papillomas or flat epithelium.

Aged↗
PubMed1981

Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation.

The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. In the present work, the uptake of rhodamine 123 by individual lymphocytes undergoing blastogenic transformation in cultures stimulated by phytohemagglutinin was measured by flow cytometry. A severalfold increase in cell ability to accumulate rhodamine 123 was observed during lymphocyte stimulation. Maximal dye uptake, seen on the third day of cell stimulation, coincided in time with the peak of DNA synthesis (maximal number of cells in the S phase) and mitotic activity. A large intercellular variation among stimulated lymphocytes, with some cells having fluorescence increased as much as 15 times in comparison with nonstimulated lymphocytes, was observed. Whereas the increased uptake of rhodamine 123 also correlated with the increase in cellular RNA content, the correlation between the dye uptake and cell size (measured by light scatter) was less apparent. As observed by UV microscopy, the increased dye uptake during the blastogenesis was due, to a large extent, to an increase in number of mitochondria per cell. However, an additional increase in rhodamine 123 binding per mitochondrion or per unit of mitochondrial membrane in stimulated cells could not be excluded. The present data indicate that rhodamine 123 may be used as a supravital mitochondrial probe, discriminating between cycling and quiescent cells and having application in sorting functionally distinct cell subpopulations.

Cell Cycle↗
PubMed1981

Effects of aclacinomycin on cell survival and cell cycle progression of cultured mammalian cells.

The effects of aclacinomycin (ACM; NSC 208734) on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent (Chinese hamster ovary) cell systems. Cell cycle progression and the effect of the drug on various transition points in the cell cycle (i.e. G1 to S phase, through a window in early S phase and G2 phase to mitosis) were monitored by flow cytometry. Formation of Chinese hamster ovary cell colonies was inhibited by 50% following 24 hr of exposure to 0.05 micrograms ACM per ml whereas 1 hr of exposure to 1.0 micrograms ACM per ml reduced colony formation by only 30%. Stationary cultures required a drug concentration more than 5 times higher to reduce colony formation by an equivalent amount when present for 24 hr. Short-term (1-hr) exposure to drug concentrations up to 1.0 micrograms/ml had no effect on colony formation of stationary-phase Chinese hamster ovary cells. Cell growth was inhibited by 50% in suspension cultures of Friend leukemia and L1210 cells when exposed for 24 hr to 0.024 and 0.053 micrograms ACM per ml, respectively. Continuous drug exposure of Friend leukemia and L1210 cells to ACM concentrations of 0.05 to 0.1 micrograms/ml led to a slow down in cell progression manifested as an accumulation of cells in G2 + M phase by 24-hr and then in G1 phase by 48-hr culture. However, brief (1-hr) exposure of L1210 cells to 0.5 micrograms/ml resulted in an irreversible accumulation of cells in G2 + M phase. A more detailed examination of drug effects on the cell cycle determined that 0.1 micrograms ACM per ml resulted in a slow down in L1210 cells leaving G1 phase and entering mitosis and an accumulation of cells in G2 phase, although early S-phase cells appeared unaffected. At a 5 times higher drug concentration, exit of cells from G1 was almost completely halted, passage of cells through early S was slowed, and the entrance of cells into mitosis plateaued 3.5 hr after addition of the drug; G2-phase cells were only mildly affected. The RNA content of all cells examined was reduced by 35 to 50% depending upon dose and time of exposure. These findings are discussed in terms of the known biochemical effects of ACM on RNA and protein synthesis.

Aclarubicin↗
PubMed1981

Effects of the L isomer (+)-1,2-bis(3,5-dioxopiperazine-1-yl)propane on cell survival and cell cycle progression of cultured mammalian cells.

The effects of the L isomer (+)-1,2-bis(3,5-dioxopiperazine-1-yl)propane (ICRF 159; NSC 169780) on cell viability, growth, and progression through the cell cycle were investigated in suspension cultures of murine leukemia (Friend leukemia and L1210) cells and normal human lymphocytes stimulated with phytohemagglutinin and in adherent cultures derived from human neuroblastoma and Chinese hamster ovary (CHO) cells. CHO cell colony formation was inhibited by 50% following either an 8.5-hr exposure of exponentially growing cells to 10 micrograms ICRF 159 per ml or a 24-hr exposure to 3 micrograms ICRF 159 per ml. This effect was cell cycle phase specific; early G1- and G2-phase cells were more sensitive than were late-G1- or early and mid-S-phase CHO cells. Stationary-phase CHO cells were unaffected by the drug at concentrations up to 500 micrograms/ml. Incubation of L1210 cells with 3 micrograms ICRF 159 per ml for 24 hr or with 10 micrograms ICRF 159 per ml for 6 hr inhibited cell growth by 50%. In contrast, 24-hr incubation of human lymphocytes with up to 50 micrograms ICRF 159 per ml had no effect on their viability or on their ability to be stimulated by phytohemagglutinin. Constant exposure of Friend leukemia, L1210, human neuroblastoma, and phytohemagglutinin-stimulated human lymphocytes to 10.0 to 50 micrograms ICRF 159 per ml resulted in inhibition of cell division which led to cell growth at higher ploidy levels. Thus, proliferating human cells of normal or tumor origin and murine leukemic cell lines all had a similar sensitivity to the drug. Detailed analysis of cell cycle progression in L1210 cells in the presence of the drug determined that cell progression through G1 phase (G1A to G1B transition) was slowed by approximately 50%. The rate of traverse of cells through S phase was also slowed. However, the most pronounced effect was the accumulation of cells in G2 phase occurring almost immediately after addition of the drug. The data suggest that the L isomer has a range of cytotoxicity and identical cytokinetic effects similar to that of the clinically tested racemate (+/-)-ICRF 159 (NSC 129943) and, therefore, that the more soluble L isomer may have increased clinical applicability.

Animals↗
PubMed1981

Multiparameter flow cytometry. Part II. application in hematology.

The application of flow cytometry to hematology is emerging as an important new technique in experimental and clinical leukemic research. In addition to the described methods to determine DNA, RNA, and esterase content and the stability of DNA in situ, quantitation of immunofluorescence using antibody techniques has greatly elucidated the hematopoietic cell system. Hence, flow cytometry provides new perspective for determining the origin, diagnosis, classification, staging and treatment scheduling of normal hematopoietic and malignant cell populations.

Aneuploidy↗
PubMed1980

Relation of mammalian sperm chromatin heterogeneity to fertility.

Flow cytometry of heated sperm nuclei revealed a significant decrease in resistance to in situ denaturation of spermatozoal DNA in samples from bulls, mice, and humans of low or questionable fertility when compared with others of high fertility. Since thermal denaturation of DNA in situ depends on chromatin structure, it is assumed that changes in sperm chromatin conformation may be related to the diminished fertility. Flow cytometry of heated sperm nuclei may provide a new and independent determinant of male fertility.

Acridine Orange↗
PubMed1980

Failure of accumulation of cellular RNA in hamster cells stimulated to synthesize DNA by infection with adenovirus 2.

AF8 cells are temperature-sensitive mutants of the cell cycle derived from baby hamster kidney (BHK) cells which arrest in the G1 phase when incubated at the nonpermissive temperature. RNA accumulation was studied in these cells by flow cytofluorimetry following serum stimulation or adenovirus 2 infection. Serum stimulation caused an increase in the amount of RNA per cell, which reached a maximum in S and G2 cells, as repeatedly reported in the literature. However, adenovirus 2 infection caused a fraction of cells to enter S phase without any concomitant increase in the amount of RNA per cell.

Adenoviruses, Human↗
PubMed1980

Bladder cancer diagnosis by flow cytometry. Correlation between cell samples from biopsy and bladder irrigation fluid.

Results of flow cytometry (FCM) examinations of bladder irrigation specimens were compared with those of FCM examinations of cell suspensions from bladder biopsies of 44 urologic patients. The fluorescent dye, acridine orange (AO), was used to stain DNA and RNA differentially and abnormal urothelial cells were identified by their relative content of nucleic acids. Granulocytes and squamous cells could be distinguished from transitional cells in this procedure, and did not interfere with the analyses. Of 28 patients with papillary carcinoma, carcinoma in situ, and invasive carcinoma 27 were identified through FCM examination of irrigation cytology specimens; the one false-negative result was from a low-grade papillary carcinoma. Of 7 patients with papilloma, FCM examinations of irrigation specimens were positive in 4 and negative in 3. Results of FCM studies of biopsy specimens were in good but not complete agreement with those of irrigation specimens. In several cases, irrigation FCM disclosed tumor stemlines that were not identified in biopsy specimens. Discrepancies of this kind seemed most likely due to differences in sampling. Irrigation FCM seems to be a sensitive method for assessing multiple-site bladder tumors, and may be a useful technique for monitoring the course of conservatively managed bladder tumors.

Acridine Orange↗
PubMed1980

Flow cytometry in bladder cancer detection and evaluation using acridine orange metachromatic nucleic acid staining of irrigation cytology specimens.

A new technique for simultaneous multiparameter deoxyribonucleic acid, ribonucleic acid and nuclear size measurements by flow cytometry was applied to the examination of bladder irrigation cytology specimens from 107 urologic patients. The cell samples from patients with bladder carcinoma could be distinguished from normal by 2 features: 1) an increase in the proportion of bladder epithelial cells with more than diploid deoxyribonucleic acid and 2) aneuploid cell peaks. These criteria identified 12 of 13 cases of invasive carcinoma, 24 of 28 cases of carcinoma in situ and 11 of 13 cases of papillary carcinoma. An increased proportion of cells with more than diploid deoxyribonucleic acid or aneuploidy was found in 9 of 14 patients with papilloma and 6 of 19 patients with a history of bladder tumors but no evident disease at present--these were believed owing to increased epithelial proliferative rates or nuclear chromatin abnormalities not visible by light microscopy. None of the 20 patients who had never had bladder tumors was abnormal. While the results in this small clinical trial have been most encouraging an additional descriptor of nuclear chromatin structure is believed necessary to discriminate benign, reactive proliferative epithelium from neoplasm when the latter is near diploid or shedding few cells. Studies to develop such a parameter presently are under way.

Acridine Orange↗
PubMed1980

Subcompartments of the G1 phase of cell cycle detected by flow cytometry.

Cellular DNA and RNA were simultaneously quantitated in individual cells of cycling and quiescent populations by flow cytometry. Based on differences in RNA content, two distinct subcompartments, G1A and G1B, were recognized in the G1 phase of exponentially growing cells. After mitosis, cells reside in the low-RNA, G1A compartment from which they cannot enter the S phase directly. An increase in RNA above a critical level is required for G1 cells to be able to initiate DNA replication; G1 cells with RNA values above this level are classified as G1B. Cell transition from G1A to G1B and from G1B to S was analyzed in a stathmokinetic experiment on L1210 cells with a doubling time of 10 hr. The half-time of cell residence in the indeterminate state of G1 was found to be 1 hr, and the duration of the deterministic portion of G1 phase was 2 hr. The indeterminate state, although not identical with G1A, is most likely located in G1A. Cell quiescence induced by serum deprivation (3T3 cells) or by addition of n-butyrate (L1210) results in cell arrest at a state which, judged by RNA content, is similar to that of G1A of exponentially growing cells. The exit from this state, however, is much slower after stimulation of these blocked cells than the transition of G1A to G1B in cells growing exponentially. Thymidine or hydroxyurea arrest cells in G1B or perhaps at the border between G1B and S. Prolonged incubation of 3T3 cells to confluence results in a marked loss of cellular RNA below the level of the G1A state. This deep quiescent state (G1Q) is distinctly different from the G1A state of cycling cells or cells blocked in G1A by serum deprivation.

Acridine Orange↗