In order to study cell proliferation in intact human cervical epithelium, a technique involving metaphase arrest has been utilized. Metaphase accumulation is observed following intraepithelial administration of vincristine sulphate, at a predetermined optimum dose of 50 μg/ml. A significant delay before the onset of stathmokinetic activity is demonstrated; thereafter linearity of accumulation is apparent over a 4-hr period following injection. The technique appears applicable to the estimation and comparison of cell production rates in vivo of normal cervical epithelium, cervical intraepithelial neoplasia, wart-affected cervical epithelium and early invasive carcinoma. However, individual values are likely to be imprecise in isolation, indicating the need to study relatively large numbers of subjects in each group. Some practical difficulties are discussed.
s of the Joint Meeting of the Cell Kinetics Society and the International Cell Cycle Society 28-31 March 1990, St Louis, Missouri, U.S.A.
Abstract. Cells growing in tissue culture as three‐dimensional, multicellular aggregates called ‘spheroids’ typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour‐like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti‐tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti‐neoplastic drugs, it is suggested that ‘drug resistance’ in tumours can be a kinetic as well as a genetic problem.
Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without (n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n = 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B4). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Gal beta 1-3GalNAc), HPA (D-GalNAc alpha) and GS I-B4 (alpha D-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia. Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/disordered proliferation.
Abstract. A model‐independent and elementary method of analysis of platelet survival is proposed. The method is based on the finding that the mean and standard deviation of the platelet lifespan can be expressed in the terms of the slope at time 0 and the area under the empirical platelet survival curve. The method is tested using Monte‐Carlo simulations and then applied to a set of clinical data.
Abstract. Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well‐established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A‐supplemented and vitamin A‐deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1 ‐9 μg/g liver, respectively, at 28 d. In contrast, in animals on the vitamin A‐deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0–32 and 0–09 μg/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d; their tracheas were depleted of retinol at 28 d. No vitamin A‐associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A‐deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild‐to‐severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A‐deficiency when the tissue levels of the vitamin are depleted.
Abstract. The present experiments with [14C]‐thymidine (TdR) and [3H]‐bromo‐deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight‐1 and that of BrdU is about 5·0 µg g body weight‐1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [3H]‐TdR doses commonly used in cell kinetic studies this proportion is only 0‐1‐1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [3H]‐TdR.
Abstract. This study was performed to determine the colony and cluster forming ability of granulocyte‐macrophage (CFU‐GM) progenitors of normal human blood low density cells cultured in a liquid culture system in the presence and absence of physiological doses of hydrocortisone (He). The CFU‐GM recovered from the liquid cultures were assayed in soft agar medium. The results of the assays indicated that time‐related development of clusters and colonies over 1‐16 days, proliferative responsiveness to a source of colony stimulating activity, number of cells developed per colony, and the cellular composition of clusters and colonies produced from CFU‐GM recovered from 14‐day‐old liquid cultures with 1·0 µM. He, were all similar to those that developed from the normal human blood low density cells. However, a higher fraction of the CFU‐GM in day 14 liquid cultures with 1·0 µM He were in DNA synthesis phase compared with the CFU‐GM from the peripheral blood. This study confirmed the results of previous studies showing lower numbers of recognizable neutrophilic granulocytes and improved survival/proliferation of CFU‐GM at day 14 in liquid cultures with 1·0 µM He compared with cultures without He. The present results suggest that the normal human blood CFU‐GM which persists and proliferates under the influence of He in a liquid culture system is similar in ontogeny to the blood CFU‐GM, and that the recovery of CFU‐GM from liquid cultures under the influence of He appears to be exerted through stimulation of proliferation and controlled differentiation.
Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.
Abstract. Progressive in vitro culturing of interleukin‐3 (IL‐3) dependent normal murine mastocytes (PB‐3) resulted in a variant cell line (PB‐1) able to grow without exogenous IL‐3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c‐myc protein and DNA content of PB‐3 and PB‐1 cells. The c‐myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB‐3 and PB‐1 cell lines, namely, the duration of the G1, S and G2+ M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse‐chase method and BrdU/DNA flow cytometry. Levels of c‐myc protein in PB‐1 cells were about twofold higher than those of PB‐3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15‐3 h for PB‐3 cells and 12‐4 h for PB‐1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2+ M phases. These results indicate that acquired IL‐3 independency in vitro and tumorogenicity of PB‐1 cells were accompanied by a doubling of c‐myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.
Abstract.Cyclic haemopoiesis in Grey Collie dogs is characterized by stable oscillations in all haemopoietic lineages. It is proposed that in these animals, in contrast to normal animals, the maturation process of haemopoietic (in particular granuloid) cells from the primitive progenitors to the functional cells is characterized by an abnormally strong synchrony. It is conjectured that the marrow maturation time has a very small variance compared with non‐cyclic normal dogs. With a mathematical model of haemopoiesis it is shown that small fluctuations are amplified via regular feedback processes such that stable granuloid oscillations are established. Erythroid oscillations are induced indirectly by granuloid feedback to the stem cell pool. The model calculations further show that the synchrony hypothesis of bone marrow maturation can quantitatively explain the following experimental results: (1) the maintenance of stable cycles of granuloid and erythroid bone marrow and blood cells with a period of approximately 14 d; (2) the disappearance of granuloid and erythroid cycles during the administration of the colony stimulating factor rhG‐CSF; (3) the reappearance of oscillations when the administration of CSF is discontinued; (4) the cessation of cycles during endotoxin application; and (5) the persistence of cycles during erythroid manipulations (bleeding anaemia, hypoxia, hypertransfusion). We therefore conclude that cyclic haemopoiesis is not caused by a defect in the regulatory control system but by an unusual maturation process.
Mitotic cells in normal, mature rat corneal epithelium were examined with a light microscope on serial, semi-thick plastic sections. Classification of mitotic figures into horizontally, obliquely or vertically positioned with reference to the epithelial basal lamina has shown that no single configuration predominates. A striking correlation between the position of the daughter cells after cytokinesis and their morphology has been observed. Horizontal cytokinetic pairs were morphologically symmetric but vertical ones were asymmetric, displaying distinct differences between daughter cells. Analysis of earlier mitotic phases has shown that the asymmetry could also be observed in vertical anaphases and telophases. The data provide clear morphological evidence for real asymmetric (unequal) cell division in a replacing epithelium in an adult mammal. It is concluded that asymmetric cell division in the corneal epithelium coexists with, and is as frequent as symmetric (equal) cell division. Randomness of mitotic spindle positioning implies that diverse forms of cell transfer from the proliferative into the differentiative epithelial compartments must operate. Therefore, the universality of the general model of cell renewal in stratified epithelia, which assumes a strong predominance of horizontal mitoses, exclusively equal mitotic divisions and one form of cell transfer, is questioned.
Abstract. The synthetic molecule N‐Acetyl‐Ser‐Asp‐Lys‐Pro (Ac‐SDKP), corresponding to the low molecular weight inhibitory factor preventing in vivo haematopoietic stem cell (CFU‐S) entry into DNA synthesis, was tested in two heterologous systems in vivo: adult regenerating rat liver and 10‐day‐old rat hepatocytes synchronized by an irritating trigger. In both systems, it was shown that doses of 2–8 μg kg‐1 of tetrapeptide inhibited 50–70% of the hepatocyte G1‐S transitions.
Cells growing in tissue culture as three-dimensional, multicellular aggregates called 'spheroids' typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour-like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti-tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti-neoplastic drugs, it is suggested that 'drug resistance' in tumours can be a kinetic as well as a genetic problem.
In some cases of acute lymphoblastic leukaemia (ALL) the percentage of cells in G2 + M is higher than anticipated when compared with the percentage in S phase. This increase in G2 + M, as detected by flow cytometry measurement of DNA content, may be due to an accumulation of cells, either in G2 or during the end of S phase; it may also be related to the existence of small tetraploid clones generally ignored by cytogeneticists. In order to identify possible subpopulations of cells with a DNA index greater than or equal to 2.0, we have compared the results of a cytogenetic analysis to the G2 + M values. We have also studied the distribution of S phase cells in 24 cases of ALL by incorporating 5-bromodeoxyuridine, labelling the cells by indirect immunofluorescence, and analysing them by flow cytometry after propidium iodide staining. The distribution of cells during S phase was quantified: no accumulation of cells was ever observed at the end of S phase. The question of the existence of small tetraploid clones, G2 arrested cells or cells with a G2 elongation remains open. However, we feel that it is more probable that, in this pathology, an elongation of the duration of G2 occurs.
Abstract. Changes in morphology and cell kinetics are described in a rat thyroid transplantable tumour (TTT) during the first few transplant generations. The growth of TTT in animals was possible only with an increased circulation level of the thyroid stimulating hormone (TSH). With serial transplantation subcutaneously in isologous animals, the morphology of TTT changed dramatically from that of a follicular tumour in the 3rd passage to become, by the 9th generation, a poorly differentiated tumour with a trabecular arrangement of cells. This change in tumour morphology was accompanied by an increase in the number of proliferating cells–mitotic index (MI), [3H]thymidine labelling index (LI), growth fraction (GF)–and cell loss factor (O) as well as a decrease in the cell cycle time (Tc) and potential population doubling time (TPD). TTT belongs to the class of tumours with a low proliferative activity and might be used in a variety of cell kinetic, radiobiological and chemotherapy studies.
The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.
Abstract. Burst‐promoting activity (BPA) was measured in the sera from 31 children with aplastic anaemia (AA). BPA levels were elevated in most of the children with A A (65‐2%), the mean value (137.7 ± 18.4%) being significantly higher than that in normal children (69.6 ± 9.4%), in children in the recovery period and in children with non‐aplastic anaemia. There was a negative relationship between the BPA level in children with AA and the peripheral haemoglobin concentration. The BPA level was higher in those whose duration of illness was shorter than 1 year. In three cases of AA caused by chloramphenicol and benzene hexachloride and one case of congenital pure red cell AA, the BPA level was not elevated. Eleven patients received fetal liver cell suspensions intravenously (FLI). After FLI the BPA level in their sera was significantly reduced. According to these results, it appears that the elevation of BPA level is a special phenomenon of A A. The measurement of BPA in serum is helpful for differentiation between AA and other kinds of anaemia. The elevation of the BPA level in serum is a biological compensation for the haematopoietic disorder, and the measurement of BPA in the serum of patients with A A may be helpful in evaluating the haematopoietic condition.
Following 15 mg kg-1 cisplatin to mice, the labelling index (LI) in the kidney decreased from 0.4% to less than 0.01% at 1-3 d, increased to 1.9% at day 16 and returned to control levels by day 30. Cytotoxicity was assessed by counts of viable tubule cross-sections and recovery was incomplete up to 14 months after treatment. Cisplatin treatment impaired the regeneration response to a dose of 16 mg kg-1 uranyl nitrate (UN) given 14 d after cisplatin when assessed by an increase in LI and sub-capsular tubule count. However, there was recovery with greater time intervals. At nine months after cisplatin there was no difference in response to UN of controls and mice previously treated by cisplatin. Prior treatment with paraplatin or iproplatin at LD50 doses produced not only no histopathological changes but also no impairment of the subsequent responses to UN.
To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2-dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9.5 mg kg-1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.
