CELL KINETICS OF A CHONDROSARCOMA

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G, 10-5 hr; S, 9-5 hr; G₂, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0–5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

Establishment and growth kinetics of the mutant Ehrlich-Lettré ascites cell strain HD33 in permanent suspension culture

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 X 10(-5) of the explanted cells continued to grow in vitro. The resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. The corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. The duration of the G1 phase was effectively zero. Both PLM data and [3H]/[14C] thymidine double-labelling measurements revealed an S-phase duration of between 11 and 12 hr. The G2 phase lasted 3-5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

Isonicotinic acid hydrazide inhibits cell population growth during teratogenesis of chick embryo

In chick embryos treated with a 4 hr pulse of 7.2 X 10(-5) M isonicotinic acid hydrazide (INH) the cell population growth is inhibited with an increased population doubling time. Teratogenised blastoderm cells complete their ongoing cell cycle and arrest in G1 phase. A chase with an equimolar concentration of pyridoxal-5-phosphate restores the growth rate after a lag of 4 hr equivalent to the duration of treatment with INH. Presumptive mesoblast cells invaginated through the primitive streak and neuroectoblast cells induced prior to the application of INH differentiate, while the teratogen inhibits morphogenesis and organization of organ primordia.     

Properties of the H-4-II-E tumor cell system. II. In vitro characteristics of an experimental tumor cell line.

The growth and cell proliferation characteristics of the H-4-II-E cell line, giving rise to hepatoma H-4-II-E when inoculated into male ACI rats, were studied in vitro. Following seedling of 2 x 10(5) cells into culture dishes, exponential cell growth occurs in cultures fed both at 24 hr and 48 hr intervals with a population doubling time of 18-4 hr. Plateau phase growth conditions are established on day 7 and day 5 for cultures fed at 24 hr and 48 hr intervals respectively. Both the plateau phase cell density and the maintenance of plateau phase appear dependent on the frequency of feeding. For cultures fed daily, the transition from exponetial growth to plateau phase results from both a reduction in the number of proliferating cells (99% v. 35%) as well as an elongation of the cell cycle (17-7 hr v. 128-4 hr). The cell proliferation characteristics of the culture are further discussed in reference to both cell growth and feeding schedules of other cell lines.     

Synchronization of Drosophila cells in culture.

We synchronized Drosophila cell lines (Schneider's line 2 and Kc) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S = 14 to 16 hr; G2 = 6 to 8 hr; M = 0.4 hr) were similar to those of Kc cells.     

STUDIES ON THE POPULATION KINETICS OF THE WALKER CARCINOMA BY AUTORADIOGRAPHY AND PULSE CYTOPHOTOMETRY

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. The transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5-6 days after transplantation, a PLM curve was performed, yielding estimates of Tc ? 18.0 hr, Ts ? 6.4 hr, T_G2+M? 4.1 hr. With the double labelling technique in vitro under 2.2 atm oxygen we obtained: Tc ? 18.2hr, Ts ? 8.2 hr, T_G2+M? 2.0hr. From pulse cyto-photometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: f_G1? (47.6 ± 1.1)%, f_s? (34.1 ± 1.0)%, f_G2+M? (18.3 ± 1.5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0.93 on the seventh and eleventh day. The cell loss factor was φ? 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.     

THE EFFECT OF ESTRADIOL ON THE CELL KINETICS IN THE UTERINE AND CERVICAL EPITHELIUM OF NEONATAL MICE

The effect of estradiol-17β on the length of the various phases of the cell cycle was studied in the neonatal mouse uterine, and cervical epithelium. A double labelling method was used, and in addition labelled mitoses were counted. In the uterus proper, estradiol shortens the length of the total cell cycle, T_c, from 17-9 hr to 15-7 hr, and the duration of S phase, T_s, from 6–7 to 5-1 hr 6 hr after estradiol treatment. 12 hr after estradiol treatment, T_c is shortened to 7-4 hr and T_s to 4–5 hr. The shortening of T_c at 12 hr is mainly due to an effect on T_G1, which is shortened from 8–55 hr in untreated animals to 1–8 hr in estradiol treated animals. The T_c of cervix epithelium cells in untreated animals was found to be 21-8 hr. After treating the mice for 6 hr with estradiol the T_c was now increased to 47 hr and further to 61 -2 hr following 12 hr treatment with the hormone. T_s increases from 8-3 hr to 15-2 hr following 6 hr estradiol treatment, and to 15-4 hr after 12 hr treatment. The effect is most pronounced in T_G1, which is lengthened from 10–95 hr in untreated animals to 28-1 hr and 43 hr, respectively, in animals treated for either 6 or 12 hr with estradiol.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

Cell cycles in the male accessory glands of mealworm pupae

During the pupal stage of Tenebrio molitor, the accessory reproductive glands of males grow by cell division. Within the secretory epithelium of the bean-shaped accessory glands (BAGs), cell numbers triple. In the tubular accessory glands (TAGs), the increase is 14-fold. There are two mitotic maxima in each gland. The first maximum occurs at 1-2 days while the second is at 4-5 days. The second maximum coincides with the major ecdysteroid peak described by Delbecque et al. [Dev. Biol. 64, 11-30 (1978)]. Nuclei were isolated from TAGs during the pupal mitotic bouts and during mitotic inactivity in the adult. After Feulgen or propidium iodide staining, the DNA content of these nuclear populations was measured by absorption cytophotometry or by fluorescence flow cytometry, respectively. The proportion of cells in each phase of the cycle was calculated using an iterative model. After mitoses have ended in the late pupa, the cells were arrested in G2. [3H]Thymidine was injected into 1- and 4-day pupae to pulse-label cells of the TAGs. After allowing various periods from 4 to 60 hr for cells to progress through G2 to reach mitosis, fractions of labelled mitoses were determined by autoradiography. From the combined cytometric and autoradiographic data, the duration of each phase of the cell cycle was calculated assuming the population was in exponential growth. Cell cycles in 4-day pupal TAGs take 48 hr. G1, S, G2, and, M lasted 13, 14, 17, and 4 hr, respectively.     

Rhodamine 123 alters the mitochondrial ultrastructure of cultured L1210 cells

Exposure of exponentially growing L1210 cells in vitro to 5-10 micrograms/ml of rhodamine 123 (R123) for 16-48 hr inhibits cell proliferation and induces cell arrest in the G1A phase of the cell cycle. The cells remain viable during the arrest and resume growth after removal of R123; extended exposure to R123 is cytotoxic. Exposure to R123 results in morphological alterations in mitochondria of all cells observed; specifically, mitochondria of R123-treated cells are characterized by a distention of the intracristal spaces and a significant increase in the number of matrix granules. Gross morphological changes of mitochondria include formation of extended organelles and the appearance of doughnut-shaped structures.     

Analysis of cell flow and cell loss following X-irradiation using sequential investigation of the total number of cells in the various parts of the cell cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. The generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G2 blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. The experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Synchronization of the human promyelocytic cell line HL 60 by thymidine

Cultures of the promyelocytic cell line HL 60 were synchronized with thymidine. A concentration of 0.05 mM thymidine and an exposure time of 24 hr was found optimal for blocking about 90% of the cells in S phase. Following release from the thymidine block the cell cultures were followed intermittently over 40 hr for fluctuation in cell numbers, labelling with radioactive thymidine and nuclear DNA distributions. Mathematical evaluation of the results revealed a cycling time of 18.6 hr and a duration of specific cell phases of 8.6 hr, 7.1 hr and 2.9 hr for G1, S and G2 + M, respectively. The doubling time was 26 hr and the growth fraction was estimated as 1.     

Flow cytometric analysis of adriamycin-perturbed mouse mammary tumors.

The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Effects of tomato paste extracts on cell proliferation, cell-cycle arrest and apoptosis in LNCaP human prostate cancer cells

Since tomato consumption is associated with decreased risk of prostate cancer, cell proliferation, cell cycle progression and apoptosis by LNCaP human prostate cancer cells might elucidate action of tomatoes. To discover possible bioactive fractions of tomatoes, whole tomato paste and its water and hexane extract were used and biomarkers of carcinogenesis were measured. After 6, 24 and 48 hr of incubation, cells were harvested and determined cell growth. Tomato paste hexane extract inhibited cell proliferation by 33% compared to the control after 48 hr incubation. Whole tomato paste and its water extract showed only modest growth inhibition. Tomato paste hexane extract at 5 microM lycopene increased G2/M-phase of the cell cycle from 13 to 28% and decreased S-phase cells from 45 to 29%. Apoptosis was observed at the 5 microM hexane extract at the late stages during 24 and 48 hr treatment. Tomato, therefore, deserves study as a potential chemopreventive/chemotherapeutic agent.     

Growth Inhibition of Helicobacter pylori by Monoclonal Antibody to Heat-Shock Protein 60

The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.     

Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line

The mechanism of the G0/G1 arrest and inhibition of proliferation by quinidine, a potassium channel blocker, was investigated in a tissue culture cell line, MCF-7, derived from a human breast carcinoma. The earliest measurable effect of quinidine on the cell cycle was a decrease in the fraction of cells in S phase at 12 hr, followed by the accumulation of cells in G1/G0 phases at 30 hr. Arrest and release of the cell cycle established quinidine as a cell synchronization agent, with a site of arrest in early G1 preceding the lovastatin G1 arrest site by 5–6 hr. There was a close correspondence among the concentration-dependent arrest by quinidine in G1, depolarization of the membrane potential, and the inhibition of ATP-sensitive potassium currents, supporting a model in which hyperpolarization of the membrane potential and progression through G1 are functionally linked. Furthermore, the G1 arrest by quinidine was overcome by valinomycin, a potassium ionophore that hyperpolarized the membrane potential in the presence of quinidine. With sustained exposure of MCF-7 cells to quinidine, expression of the Ki67 antigen, a marker for cells in cycle, decreased, and apoptotic and necrotic cell death ensued. We conclude that MCF-7 cells that fail to progress through the quinidine-arrest site in G1 die. J. Cell. Physiol. 176:456-464, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of growth of ASL-1w murine leukemia cells by anti-thymus leukemia antigen (TL) serum in the absence of complement.

Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.     

Cell cycle changes during neural crest cell differentiation in vitro.

Chick trunk neural tubes containing neural crest cells were cultured in vitro. Cell outgrowth from these neural tube explants consists primarily of a small stellate cell population. After 3 days in culture the small stellate cell population undergoes a remarkable change in morphology that is characterized by a more refractile appearance in the phase contrast microscope. Subsequent to this change in morphology, pigment granules become visible in the cytoplasm after 4 days in culture. After 6 days in culture, virtually all of the small stellate cells are pigmented. The cell cycle parameters of the small stellate cell population are: S = 4.4 ± 1.2 hr (SD). G2 = 1.5 ± 1.0 hr (SD). M = 1.7 ± 0.6 hr (SD). and Gl = 3.8 ± 1.0 hr (SD). Continuous label experiments demonstrate that (G1+G2+M) increases from 7 hr in Day 4 cells, as yet unpigmented, to 12 hr in Day 5 cells that have become pigmented. This change is consistent with an increase in G1 and/or G2 that is closely correlated with the appearance of pigment granules. It is of interest that this cell cycle change is correlated with a rather late event in the developmental program of these neural crest cells rather than with the earlier morphological change observed after 3 days in culture.