Transformation of human embryonic cells by the Rous sarcoma virus and the polyomavirus depending on the mitotic cycle phase]

The whole cycle of skin-muscle embryonic human tissue culture is 18 hours, with phases S, G1, G2 and M being 7, 6, 4 and 1 hour, respectively. The mitotic index of this culture is 28%. The maximum sensitivity of these synchronized cell cultures to transforming activity of the Rous and Sindai viruses was observed in phase S. The infection of synchronized primary embryonic human fibroblasts in phase S with the polyoma virus together with the Sindai virus has resulted in single cases of transformation. Similar results were obtained with non-synchronized human cultures.     

Effect of hydrocortisone on the mitotic cycle of epithelial cells in the mouse pylorus]

It was shown with the aid of thymidine-H3 that the mitotic cycle of mucous-forming cells (superficial epithelial mucosal cells of the neck) of the stomach pyloric glands of mice lasted 13.5 hrs (G1+1/2M = 7.6 hrs, S = 5.3 hrs; G2+1/2M = 0.6 hrs). With the administration of a physiological dose of hydrocortisone (0.1 mg) the duration of the mitotic cycle of mucous-forming cells of the stomach pyloric glands increased by 6.7 hrs (G1+1/2M = 11.6 hrs, S = 7.8 hrs; G2+1/2M = 0.8 hrs). A high dose of the hormone had a similar effect and increased the presynthetic period to 12.9 hours and the postsynthetic one--to 2.3 hours.     

Regenerative growth of asvitic hepatoma 22A]

A study was made of the recurrent growth of ascite hepatoma 22A, occurring at transplantation of 11-12-day old tumours to new hosts. The mitotic activity of the hepatoma was found to increase by 3 to 4 times (12-15 hours after inoculation). This increased cell proliferation is due mainly to a sharp shortening of all the periods of mitotic cycle. During the recurrent growth, the resting R1 cells resume their mitotic cycle. No resumption of the mitotic cycle by the resting R2 cells was observed     

TIMING OF DNA SYNTHESIS IN THE MITOTIC CYCLE IN VITRO

A study was made of the timing of DNA synthesis in the mitotic cycle under conditions where the average mitotic cycle of populations of human amnion and kitten lung cells in culture was variable. Three types of experiments were performed: (a) Autoradiographs were made of incorporated tritiated thymidine in cells whose mitotic histories were recorded microcinematographically allowing the measurement of telophase + G1 along with the total length of the mitotic cycle. (b) Measurement of the G2 + prophase part of the mitotic cycle was performed under various conditions by exposing cells to tritiated thymidine and observing the increase in labeled metaphases plus anaphases as a function of time. (c) The effect of a change in pH on parts of the mitotic cycle was tested by continuously photographing a single colony of cells first at pH 7.8 and then at pH 7.1. All of our data point to the same conclusion; namely, that within a population of cells with a given generation time, the length of each of the measurable parts of the mitotic cycle has a particular distribution of values and that, when there is a change in the generation time, under our conditions only the T + G1 distribution changes.     

Cell population kinetics and the cell population mechanisms of the circadian rhythm of proliferative activity in mouse esophageal epithelium]

The dynamics of 3H-thymidine labeled mitosis and diurnal rhythm of proliferative activity was studied. The isotope was injected to BALB/C mice at the peak of diurnal rhythm of DNA synthesis activity of basal layer cells of oesophageus epithelium. It has been established that the increase in the mitotic index during 24 hours depends on the increase in number of cells being in S-period. The data show that the increase of mitotic index at diurnal rhythm occurs at the expense of 75% of new G0-cells which entered into the mitotic cycle, and of 25% of re-entering cells that had divided during the maximal mitotic activity a day before. It is found that the duration of mitotic cycle of cell population which entered into the mitotic cycle synchronously is almost equal to the period of diurnal rhythm of mitotic activity, i.e. 24 hours.     

Immediate effects of serum depletion on dissociation between growth in size and cell division in proliferating 3T3 cells

Proliferating nonconfluent 3T3 cells become committed to proceed through the cell cycle or to enter G0 during the first post-mitotic part of G1 (G1pm). The decision to proceed through G1pm is dependent on the presence of serum growth factors in the culture medium. Cells that have passed this particular growth-factor-dependent cell cycle stage are independent of serum growth factors and undergo mitosis on schedule. We report here that G1ps, S, and G2 cells cease to increase in size when serum is withdrawn. As a result the mitotic cell size after 8 hours serum starvation is reduced to approximately 60% of the normal mitotic cell. This reduced growth in cell size is due to a rapid decrease in protein synthesis and some increase in protein degradation. This dissociation between growth in size and cell-cycle progression within a single cell cycle provides a new approach to study the two processes separately.     

Study of the effect of cAMP on the mitotic regimen in the esophageal epithelium of tumor-bearing mice]

The mitotic activity and number of DNA-synthesizing cells in the epithelium of the esophagus of the tumour-bearing albino mice were studied for 24 hours after the injection of dibutyryl cyclic 3', 5'-AMP. It was shown that injection of the preparation led to the blocking of cells in the G2-phase of the mitotic cycle, and to prolongation of mitosis during the first hours of the experiment without changing the total number of cells undergoing mitosis in the course of 24 hours.     

Effect of 2-mercaptoethanol on the individual periods of the mitotic cycle

Dynamics of the mitotic cycle of the KEPV cells being on different interphase stages at the start of a 20 hour 2-mercaptoethanol (0.001 M) treatment has been studied during the treatment and for 11 hours after washing out the agent. The KEPV cells affected by mercaptoethanol during the interphase (G1, S, G2) were shown to continue their passage through the cycle to enter mitosis, but part of the cells of the S period and of the first half of the G2 period were arrested in the interphase. In the presence of mercaptoethanol, mitotic cells reach the metaphase stage, and their further behaviour depends on the duration of the treatment. For the first 8 hours of treatment, a phase of "unstable block" exists for cells that were in S and G2 periods at the beginning of treatment, while other cells are transformed into K-metaphases. 8 hours later a phase of "stable block" occurs and all the normal metaphases are transformed into K-metaphases. After washing out the culture from mercaptoethanol the cells are ejected from the block in K-metaphase. The transformation from K-metaphase into the normal metaphase is realised in the course of this process. The cells which were in S and G2 periods at the beginning of the treatment are ejected from the block simultaneously after washing, while the cells of the G1 period--with a small delay. After washing out mercaptoethanol the cells that were in the interphase (G1, S, G2) at the beginning of the treatment are capable of producing both multipolar mitoses and mitoses without cytotomy.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

Circadian rhythm of hepatoma 22a cell division after exposure to liver chalone

Chalone isolated from the cells of the normal rat liver exerts a pronounced inhibitory action on hepatoma 22a cell division during 9 hours after its administration. Then the mitotic activity of hepatoma cells returns to the control level, but after 15 hours it starts to diminish again. The total amount of cells that entered the mitotic cycle during the experiment declined by 30% as compared to the control. Thus, hepatic chalone produces a reversible inhibition of hepatoma cell division in G2 and G1 phases of the mitotic cycle and lessens the fraction of dividing cells over a period of 24 hours.     

Effect of the time before fixation on the yield of chromosome aberrations in a culture of human lymphocytes exposed to gamma rays at different stages of the mitotic cycle

A comparative study was made of the yield of chromosome aberrations in human lymphocyte culture exposed to 60Co-gamma-rays (2 Gy) at different mitotic cycle stages the cells being fixed after 52 and 60 hr. It was shown that with the latter fixation time (60 hr) the frequency of chromosome aberrations after irradiation in G1 stage was substantially lower than that with the former one (52 hr) and, vice versa, it was higher after irradiation in S and G2 stages. The authors discuss the probable causes of the distinctions observed.     

Rate of progress of the 1st post-stimulation division of the mitotic cycle by cells of ascitic hepatoma 22A of different ages]

A study was made of the progress rate of cells of the ascitic hepatoma 22A of different age during the iirst mitotic cycle after the stimulation of division. The "ageing" (11-day), terminal (14-day), and "delayed" (4 days older than the terminal stage) ascitic fluids were used. The maximal values of the labeled nuclei index was found to be reached by 9--12 hours (it was mainly due to the transtion of the quiescent to the S-period) and the maximal mitotic index--by 18--21 hours after the inoculation, independently of the tumour age. These results suggest that the duration of both the prereplicative (G1) period and of the whole first mitotic cycle after the stimulation were independent of the time during which the cells of the ascitic hepatoma 22A were at the resting stage or at the very prolonged G1-period.     

De-regulation of the RBBP6 isoform 3/DWNN in human cancers

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.     

Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements

Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 x 10(-11) M and approximately 76,500 receptors/cell. Neither EGF nor TGF-alpha were mitogenic for NFMs, and TGF-beta reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2/M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.     

Membrane transport in synchronized Ehrlich ascites tumor cells: uptake of amino acids by the A and L system during the cell cycle.

Using the double thymidine block technique. Ehrlich ascites tumor cells (ELD) carried in continuous spinner culture have been synchronized. Simultaneous monitoring of 3H-thymidine incorporation, cell number and mitotic index yielded a cell cycle time of approximately 13.5 hours. This is composed of an S period of 3-4 hours. G2 of 6-8 hours and M of 1-2 hours. No appreciable G1 is present. Ehrlich cells synchronized in this manner were used to investigate the characteristics of two neutral amino acid transport systems during progression through the cell cycle. Unidirectional influx via the Na-dependent system A was studied using C14-alpha-aminoisobutyrate (AIB) as substrate. The Na-independent system L was monitored using 3H-leucine and 14C-cycloleucine as substrates. Transport by the A system was minimal in M and early S. It underwent a three-fold increase during late S and early G2. In mid G2 the transport via this system rapidly dropped and remained low again through M and early S. The intracellular/extracellular ratios of AIB indicate that the system is actively transporting AIB thoughout the cell cycle. The minimum ratios of approximately 3 were achieved during early M and the maximum ratios of approximately 9 were achieved in late S, early G2. The uptake of leucine and cycloleucine by the L system was quite different during the cell cycle. Maximal unidirectional influx by this system occurred during early and mid S period. Upon progression into G2 the transport rate dropped and remained reduced throughout M. Intracellular/extracellular ratios of leucine or cycloleucine were near unity at the peak of the transport activity (early S) and dropped to values of 0.5 to 0.6 throughout the remainder of the cycle. This result indicates that inward transport by the L system is, for the most part, non-active in growing cells.     

Circadian rhythm of the mitotic activity and of the number of nuclei synthesizing DNA in sarcoma 37 in white mice]

Circadian variations in the frequency of mitoses and the number of nuclei labed with thymidine-H3 in sarcoma-37 of mice were investigated. It was shown that the circadian rhythm of mitotic activity was composed of diurnal variations in the frequency of labeled and unlabeled mitoses. The G2-phase of mitotic cycle of the cells with labeled mitosis was approximately one hour. The G2-phase of the cells with unlabed mitosis lasted four hours and more. It is suggested that there are two cell populations in sarcoma-37.     

Mechanism of action of interferon is linked to its ability to protect the cell from interstrand cross-linking of DNA, induced by 8-methoxypsoralen, activated by long-wave UV-light

Treatment of human lymphocytes in the G1 phase of mitotic cycle with human lymphoblastoid interferon (Ly-IFN) decreased the frequencies of chromosome aberrations induced by 8-methoxy-psoralen-induced interstrand cross-links. Anticlastogenic effect of Ly-IFN was accompanied by stimulation of unscheduled synthesis of DNA in the G2 phase of mitotic cycle, as shown by increased percent of labeled cells registered by 3H-thymidine autoradiography. The data obtained seem to indicate that the mechanism of Ly-IFN protection is connected with stimulation of postreplicative repair.     

Action of the chalone from Ehrlich's ascites, tumor in mice on the mitotic activity in this tumor after single and double administrations

Chalone from Ehrlich's ascites tumour exerts a short-lived and reversible inhibitory effect on cell proliferation in the tumour both after a single and two-fold administration. 10 hours following single and two-fold injection of chalone (second injection was given at 6 p.m.), the mitotic index in tumour cells rises as compared to controls an evidence of chalone action on G(2) cell population of the mitotic cycle and synchronization of cell division. Repeated injection of chalone at 9 p.m. results in a more prolonged effect on the cells and in a more pronounced synchronization wave of G(2) cell population comparatively to its injection at 6 p.m. Thus the duration of cell inhibition in G(2) phase of the mitotic cycle depends with repeated administration of chalone, on the condition of cell population affected by chalone.     

CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G₁, S, and G₂ periods of interphase.¹ The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours.     

The in vitro response of thymic lymphocytes of the pig to phytohemagglutinin

The response of thymic lymphocytes of the pig to phytohemagglutinin was studied with H³ thymidine in cultures, from 0–72 hours. At the beginning of the culture period 6–18% of lymphocytes were in DNA synthesis. during the first 24 hours a sharp decrease in the number of DNA synthesizing cells was observed in both pha and control cultures, although pha cultures consistently showed small but significantly greater numbers of DNA synthesizing cells. this was followed by a definite peak in DNA synthesis and mitotic response of a minority of the cells in pha cultures between 48–54 hours, whereas in control cultures activity ceased. in addition, a small proportion of the progeny of initially DNA synthesizing medium sized lymphocytes was apparently stimulated by pha and found in mitosis by 48 hours. It was concluded that the thymus contains a fraction of lymphocytes, not in the mitotic cycle, which are capable of being transformed by pha to mitotic activity. the data also suggests some stimulation of cells already in the mitotic cycle.