Cell and tissue kinetics最新文献

We reported previously that deoxycytidine (CdR) enhances the cytotoxic effects of the drug combination thymidine (TdR) plus 5-fluorouracil (FUra) against HeLa S-3 cells. We have now examined the relationships between the concentration of CdR and its cytotoxic and cytokinetic effects, and have also investigated the role of certain other components of the culture medium in this phenomenon. Cell survival was determined by a colony-forming assay; cytokinetic effects were monitored by flow cytometry. In the initial experiments, cells were grown in Ham's F12 medium and exposed for 22 hr to 4 mM TdR, 0 X 025 mM FUra, and dCyd ranging from 1 microM to 4 X 0 mM. The individual drugs were at most only slightly toxic under these conditions; for TdR plus FUra, the survival decreased to 50% (in 5% FCS), and in the three-drug combination it varied from 8% at 1 microM CdR to 28% at 0 X 10 mM and back to a low of 3% at 4 X 0 mM CdR. Results from flow cytometry appeared correlated with the survival data, in that cells accumulated in the S phase to a greater extent in the region around 0 X 10 mM CdR than at higher or lower concentrations. When cells were exposed to the drugs in MEM medium in place of F12, their sensitivity to FUra and the TdR-FUra combination was enhanced, although the additional synergistic effect of CdR was reduced. We found that hypoxanthine, present in F12 but not in MEM, was the principal compound responsible for the observed differences between media.

This study was designed to investigate the mitotic response to wounding in the rat thyroid. The spatial distribution of mitotic activity 48 hr after incision of the thyroid isthmus, or mere exposure of the gland (sham-operation), was assessed using a stathmokinetic technique. Incision resulted in a 66-fold increase over normal in metaphase index adjacent to the wound, falling over 2 mm to a stable 13-fold elevation. Sham-operation produced a smaller response with a complete return to normal levels over 1-1 X 5 mm. The results demonstrate that there is a dramatic localized mitotic response to wounding in the thyroid together with a smaller generalized response. Further, the response to sham-operation indicates that thyroid follicular cells respond to a diffusible 'wound hormone'. We suggest that this may be a major mechanism mediating reparative growth in this gland.

Using radioautographic smear preparations of thymocytes and mesenteric lymph node (MLN) cells labelled with three different tritiated pyrimidine deoxyribonucleosides, the incorporation of DNA precursors was studied separately on large lymphocytes and small lymphocytes. Radioautographic reaction due to generally tritiated deoxycytidine ( [G-3H]CdR) labelling in vivo in large lymphocytes was more intense than that in small lymphocytes. When mice were sacrificed 6 hr after the administration of tritiated thymidine ( [3H]TdR), small lymphocytes were labelled more heavily than large lymphocytes. However, labelling intensity with [3H]TdR in large lymphocytes was greatly enhanced by the administration of 5-fluoro-deoxyuridine, whereas in small lymphocytes labelling intensity was only fairly enhanced by the same treatment. When cells were incubated in vitro with 5-tritium labelled deoxycytidine [( 5-3H]CdR) for 10 min, there was no significant difference in labelling intensities between large and small lymphocytes. In the case of [G-3H]CdR incorporation, the labelling intensity in large lymphocytes was found to be significantly stronger than that in small lymphocytes. Large as well as small lymphocytes incorporated [3H]TdR very well in vitro. However, addition of 5 X 0 X 10(-5) M of non-radioactive CdR to the medium greatly decreased the incorporation of [3H]TdR by large lymphocytes, whereas the effect of non-radioactive CdR in small lymphocytes was not so marked as that in large lymphocytes. Furthermore, the [3H]TdR-labelling percentages were decreased at the same rate by the addition of non-radioactive CdR in both large and small lymphocytes. These results indicate that large lymphocytes and a proportion of small lymphocytes have a strong tendency to convert CdR to thymidine mono-phosphate, which is utilized for DNA synthesis, whereas this ability is relatively weak in the rest of small lymphocytes. Thus, it is probably that this metabolic ability changes during the transition of the large lymphocyte to the small lymphocyte.

Previous studies have indicated that the generation time of human leukaemic cells is longer than that of normal haematopoietic cells. We have employed a modification of the thymidine (TdR)-suicide technique to measure directly the generation time of leukaemic progenitor cells capable of colony formation. The results obtained with two human leukaemic cell lines (KG-1 and HL-60) and with blast progenitor cells from two patients with acute myelogenous leukaemia indicate generation times ranging from 9 X 0-22 X 0 hr and S-phase durations ranging from 5 X 5-8 X 0 hr. Using the same technique, the generation time of normal bone marrow CFU-c was determined to be 9-11 hr. These findings suggest that the proliferation rate of human leukaemic blast progenitor cells is similar to that of normal haematopoietic stem cells.

The right cheek pouch of hamsters was painted twice a week with DMBA for 4 and 7 weeks in different groups. It was found that there were diurnal rhythms and non-random distributions of DNA synthesis and mitotic events in DMBA-treated hamster-cheek-pouch epithelium. The degree of these changes was markedly greater than the changes in non-treated control pouches. The finding of cluster formation was described as 'symphokinesis', the non-random grouping of basal cells with synchronous DNA cycles during tissue growth.

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.

The effect of thymus deprivation on spleen colony-forming unit (CFU-s), the erythroid repopulating ability (ERA) of bone marrow cells, erythropoietin-responsive cells (ERC) and the erythroid colony-forming unit (CFU-e) was investigated in adult mice. Female CBA mice, thymectomized at the age of 6 weeks, were used for the experiments at 3 to 5 weeks later (Tx). Thymectomized, lethally irradiated and bone marrow cell-reconstituted mice (TIR) were used 2 months after reconstitution. A decrease of ERA was found if Tx animals were used as recipients of normal or Tx bone marrow cells. Estimated values of ERC and number of CFU-e-derived colonies in the bone marrow and spleen in response to 3 units of erythropoietin (Ep) were lower in Tx than in control polycythaemic mice. In TIR animals, a lower number of femoral CFU-s was found, but the ERA and ERC values were not affected. However, in response to 3 units of Ep, the number of CFU-e-derived colonies differed in the bone marrow of TIR animals as compared to the values found in normal, reconstituted mice.

Mouse mammary adenocarcinoma Ca 755 was studied at two times in its growth: the exponential (8 days) and plateau phase (20 days). Cycling cells were labelled with [3H]thymidine injections at 4-hr intervals over 72-hr periods, i.e. three to five times longer than the generation times for the twentieth day and eighth day tumour cells respectively. By autoradiography, the increase of non-cycling cells in ageing tumours was confirmed. By single cell cytophotometry used after Feulgen staining it has been shown that the cells with a high DNA content (especially a G2-DNA amount) were in a higher proportion in the twentieth day tumours than in their eighth day counterparts. Combined cytophotometric and autoradiographic procedures have shown that nearly all cells with a G2-DNA content entered a non-cycling state in ageing tumours.

Cell kinetic parameters measured by the percentage labelled mitoses (PLM) technique have been compared with those derived from continuous labelling and stathmokinetic data in the mouse tumour Carcinoma NT. In [3H]TdR labelled tumours, a PLM curve showed the length of DNA synthesis (TS) to be 9 hr. With continuous labelling, 2.5% of cells entered S phase per hour and TS was 16 hr. Six per cent of cells incorporated [3H]TdR between 1 and 3 hr after an injection of the label. The stathmokinetic technique showed that the rate of entry to mitosis was 2.0% hr. The turnover time (TT) of the population was found to be 16 hr with the PLM data and 35 and 37 hr with the continuous labelling and stathmokinetic data, respectively. The PLM technique therefore showed the mean cell cycle parameters to be about half the values obtained with other methods of measurement. We conclude that in Carcinoma NT there is a wide range of cell cycle times. The continuous labelling and stathmokinetic data represent the mean cell cycle parameters, while the PLM data give the minimum values. A cell loss factor calculated from the PLM data would, therefore, be too high by a factor of two.

Increased cellular polyamine levels are thought to be essential for epidermal keratinocyte proliferation. However, a number of studies report that the induction of keratinocyte proliferation and of ornithine decarboxylase, the rate-limiting enzyme of putrescine, spermidine and spermine biosynthesis, is not concordantly expressed. The relationship between epidermal keratinocyte polyamine synthesis and proliferation was studied in neonatal mouse keratinocyte cultures using specific inhibitors of ODC activity to decrease the intracellular polyamine levels. The ODC inhibitors alpha-methyl ornithine (alpha-Me-Orn), alpha-hydrazino ornithine (alpha-HO) and difluoro-alpha-methylornithine (alpha-DFMO) did not significantly inhibit epidermal keratinocyte proliferation at 5 X 10(-3) to 10(-4) M concentrations. At these doses, only alpha-DFMO was seen to decrease (by 70%) the cellular levels of putrescine, but not of spermidine or spermine. Epidermal keratinocyte growth in the higher dose of 20 mM alpha-DFMO, however, did not decrease the cellular levels of putrescine. Polyamine analyses of the spent medium showed that growth in 10 mM alpha-DFMO decreased the normal epidermal cell transport of putrescine and spermidine into the medium. At 20 mM alpha-DFMO concentration, the keratinocytes actually transported, intracellularly, the putrescine and spermidine that are naturally found in the foetal bovine component of the growth medium. We conclude from these studies that epidermal keratinocyte polyamine levels are determined by both the rate of synthesis, and of the transport of these amines into the extracellular medium. Since epidermal keratinocytes actively maintain specific polyamine levels, it appears that these molecules are essential for epidermal keratinocyte function.