The cell kinetics of the adaptation of the human esophagus to organ culture

Summary The adaptation of normal human esophageal explants to organ culture for the first 33 d of in vitro growth was evaluated using histomorphology and [³H]TdR autoradiography combined with mitotic blockade. On the 3rd d in culture, extensive desquamation of superficial cells reduced the epithelium to about four cell layers. Thereafter, the epithelium remained atrophic, with a relative increase in basal and suprabasal cells. The percentage of cells synthesizing DNA was greatest from Day 4 through 8, just after desquamation, and reached a maximum on Day 4 (24 h [³H]TdR labeling index of 62%). The labeling index (LI) fluctuated, thereafter, but remained high (26% on Day 33). During the last 6 h of each [³H]TdR labeling interval, mitosis was blocked by colcemid. The 6 h mitotic rate (MR) was a reasonably constant fraction of the LI (maximum at 4 d: MR=1.44%), but was much lower than predicted by [³H]TdR labeling indicating the loss of large numbers of cells after DNA synthesis but before or during mitosis. Unlabeled mitotic figures appeared between Days 1 to 3 and 6 to 33, suggesting that the epithelium initially contained a considerable population of cells arrested or delayed in G₂ and continued to generate cells that remained in premitosis longer than 24 h. These results indicate that the atrophy observed in vitro is characterized by a relative increase in the basal and suprabasal cell category, a high replication rate, initial recruitment of cells arrested in premitosis, and rapid cell turnover with significant loss of cells at the premitotic or mitotic step, or both. Thus it seems that human esophageal epithelium grown in organ culture is a satisfactory substrate for experimentation (for example, in vitro carcinogenesis) that requires cell replication. However, there are major differences between the kinetics of esophageal epithelium in vivo and in vitro. Supported in part by Contract NOI-CP-75909 and NOI-CP-25604-59 from the National Cancer Institute, Bethesda, MD.     

Age-related changes of epidermal cell kinetics in the hairless mouse

Cell kinetic variables in normal untreated hairless mice were studied in order to observe possible age-related changes. Generally, groups of 4 male and 4 female mice were subjected to study at various ages from one to 115 weeks. The number of basal and suprabasal cells per microscopic field was observed, and after injection of tritiated thymidine the mean labelling index, the average specific activity and the mean grain count were scored. After injection of Colcemid, the average number of Colcemid-arrested mitoses was counted. With flow cytometry the fraction of cells in S and in G2 + M was also observed. In general, both the number of suprabasal cells and the proliferative variables were significantly lower in the very young mice. They increased to slightly above normal values at about 20-22 weeks of age, and then fluctuated a little with two additional possible peaks at 40-50 and around 80 weeks, respectively, and two troughs some weeks after the peaks. However, this rhythmicity was slight and not significant. Thus the only significant age-related pattern was that very young mice have a thin epidermis and low proliferative variables. These values increase up to the age of 20 weeks, and from then on there are no obvious and significant alterations, only slight rhythmic undulations almost within normal limits. The importance of cell kinetic changes with age for epidermal carcinogenesis is discussed in relation to these observations.     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

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.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

G2 sub-population in mouse liver induced into mitosis by lead acetate

A single intracardiac dose of lead acetate (40 microgram lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. The prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G2 phase. The injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. The incremental labeled cells may have originated from a small fraction of the G1 population that was ready to enter the S phase without the usual pre-synthetic delay.     

G2 Sub-Population In Mouse Liver Induced Into Mitosis By Lead Acetate

A single intracardiac dose of lead acetate (40 μ lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. the prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G₂ phase. the injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. the incremental labeled cells may have originated from a small fraction of the G₁ population that was ready to enter the S phase without the usual pre-synthetic delay.     

Turnover and maturation kinetics in the hairless mouse epidermis. Continuous [3H]TdR labelling and mathematical model analyses

Hairless mice were continuously labelled with 10 microCi of tritiated thymidine ([3H]TdR) every 4 h for 8 d, and the proportions of labelled basal and differentiating cells were recorded separately. The mitotic rate was measured by the stathmokinetic method and the cell cycle distributions were measured by flow cytometry of isolated basal cells at intervals during the labelling period. The mitotic rate of the [3H]TdR-injected animals did not deviate from control values during the first 5 d. Computer simulations of the data based on various mathematical models were made, and three main conclusions were obtained: (1) a large spread in transit times through the G1 phase was found, together with a very narrow distribution in maturation time of differentiating cells; (2) about 20% of the differentiating cells were estimated to leave the basal cell layer directly after mitosis. This is consistent with results obtained from different sets of data; and (3) during continuous labelling more than 90% of the cells are labelled during each passage through the S phase.     

CELL KINETICS OF IRRADIATED EXPERIMENTAL TUMORS: CELL TRANSITION FROM THE NON-PROLIFERATING TO THE PROLIFERATING POOL

Parenchymal tumor cells of murine mammary carcinomas can be divided into two pools, using nucleoli as morphological ‘markers’. Cells with dense nucleoli traverse the cell cycle and divide, thus constituting the proliferating pool. Cells with trabeculate or ring-shaped nucleoli either proceed slowly through G₁ phase or are arrested in it. The role of these non-proliferating, G₁ phase-confined cells in tumor regeneration was studied in vivo after a subcurative dose of X-irradiation in two transplantable tumor lines. Tumor-bearing mice were continuously injected with methyl[³H]thymidine before and after irradiation. Finally, the labeling was discontinued, mice injected with vincristine sulfate and cells arrested in metaphase were accumulated over a 10-hr period. Two clearly delineated groups of vincristinearrested mitoses emerged in autoradiograms prepared from tumor tissue at the time of starting tumor regrowth: one group with the silver-grain counts corresponding to the background level, the other with heavily labeled mitoses. As the only source of unlabeled mitoses was unlabeled G₁ phase-confined cells persisting in the tumor, this observation indicated cell transition from the non-proliferating to the proliferating pool, which took place in the initial phase of the tumor regrowth. Unlabeled progenitors have apparently remained in G₁ phase for at least 5–12 days after irradiation.     

Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells

Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20- 24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [3H]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients. Synthesis and secretion of light- chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after motosis the cells which then entered the G1 phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm^-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 /iCi tritiated thymidine ([³H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [³H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [³H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [³H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Effects of hydroxyurea on the mitotic activity and the G2 phase in hairless mouse epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid at various times after HU. The animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G2 was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G2 for the first 6 hr after HU injection, then the G2 compartment was emptied. The results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G2 and M. The cell kinetic effects of HU thus seem to be very complex.     

Sister chromatid exchanges and lack of isolabeling in Chinese hamster chromosomes

Chinese hamster CHO and Don cells were synchronized in mitosis with or without a Colcemid treatment, labeled with ³H-TdR through an entire cell cycle, and were synchronized again each time they entered mitosis. Over four cycles without label, only heterolabeling patterns were observed, and the frequency of sister chromatid exchanges (SCE's) per chromosome per cycle (0.23 or 0.02/μm) was constant. After the labeling cycle, during which the total SCE frequency was also 0.23, a disproportionately high SCE frequency at the centromeres, about 0.15, decreased dramatically to about 0.03, while the SCE frequency in the arms increased from about 0.08 to 0.20. In accord with reports in the literature, the constant SCE frequency of 0.23 appeared to be the saturation frequency resulting from the ³H disintegrations. It was postulated that during the labeling cycle, the limited number of SCE's occurred preferentially at the centromere because ³H disintegrations occurring in the daughter strand of DNA produced alterations which were preferentially repaired by a recombinational SCE mechanism. Implicit in this hypothesis is the assumption that SCE's result from the transmutation or recoil effect of the ³H disintegration and that there is a unique association between the kinetochore and the daughter strand of DNA.     

A CHARACTERIZATION OF THE BOUNDARY BETWEEN THE CYCLING AND RESTING STATES IN ASCITES TUMOR CELLS

Growth deceleration of an Ehrlich ascites tumor with increasing mass is associated with a prolongation of the cell cycle and a decline in the growth fraction. These effects are reversed upon transfer of cells from an older tumor into a new host. Studies were made to locate the stages at which a cell cycle could be suspended or resumed. Transplantation caused a prompt rise in both mitotic and flash H³TdR labeling indices. When all the cells in cycle including mitoses were prelabeled with H³TdR in older tumors, the fraction of labeled mitoses did not decline for a considerable period after transplantation into new hosts. This suggests that the early rise in mitoses is not due to a flow of resting (G_o) cells from a G² store (G²-G^o transition). It appears rather to be a reflection of a lag of the mitotic process relative to other stages during the initial readjustment of the cycle. A prompt rise in flash H₃TdR indices in the transplants suggested cell entry into S from either a suspended G_I (G₁-G_o transition) or a suspended S (S-G_o transition). These possibilities were examined by relating micro-spectrophotometric estimates of DNA to the cell cycle stage as revealed by H³TdR autoradiography. Since G_o cells had DNA values corresponding to G_I, it was concluded that decycling or recycling could occur only after mitosis and before DNA synthesis.     

RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid

To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid-induced denaturation) were studied in uniform-age mitotic CHO cell populations maintained either at 37 degrees C (in the presence of Colcemid) or at 0-4 degrees C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37 degrees C with Colcemid or at 0-4 degrees C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

The present experiments with [14C]-thymidine (TdR) and [3H]-bromodeoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 microgram g body weight-1 and that of BrdU is about 5.0 micrograms 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.     

Mitotic polyploidization of mouse heart myocytes during the first postnatal week

Summary Polyploidization of myocytes in the cardiac ventricle of mice occurs predominantly during the first week of the postnatal life. Using isolated cells it was shown that about 70 % of the cardiomyocytes become binuclear at this time (2c×2), while 10 % are mononuclear but contain 4c of DNA, where c was the haploid level of DNA. About 2 % of the cell population were represented by 4c×2 or 8c cells.Cytophotometry of Feulgen-stained DNA in ¹⁴C-thymidine-labeled nuclei has shown that the cells that enter the mitotic cycle are mainly diploid. After mitosis (30 h or more after thymidine application) the label was found predominantly in 2c×2 and 4c cell types. Comparison of the curves presenting dynamics of labeled mitoses and the accumulation of labeled binuclear cells reveals that binuclear 2c×2 cells are formed by acytokinetic mitosis. The formation of 4c mononuclear cells is accomplished via other types of mitotic arrest; this may be due, for example, to a block in the pro-or metaphase.Only very rare cases of cytotomy were detected and the number of newly formed 2c cells was very low. It is concluded that cell multiplication is practically arrested at this period of life, and growth of the ventricular mass is due to polyploidization of virtually all cycling cells, while their number remains unchanged. Mechanisms and functional significance of cardiomyocyte polyploidization are discussed.     

秋水仙胺(colcemid)诱发雄性小鼠生殖细胞减数分裂延迟和非整倍体的研究

本文以101/E1和C3 H/E1的杂种第一代雄性小鼠为材料,一次性腹腔注射秋水仙胺(COM)(1mg/kg体重)后,分别于第2,6,10,14和18h取材,进行减数分裂延迟和中期Ⅱ(MMII)染色体分析。结果表明,COM可诱发小鼠减数分裂延迟,并对其可能的生物学机制进行了讨论。在本实验条件下,COM未能导致MMII非整倍体率显著升高,并对可能的原因进行了分析。     

Occurrence of premature chromosome condensation in mouse spermatogonia treated with busulfan

Busulfan induction of premature chromosome condensation (PCC) was examined in mouse spermatogonia. Adult (C3H X SWV) F1 male mice were injected intraperitoneally with busulfan at 10 or 30 mg/kg of body weight and killed 18-72 h later. Polyploid-like mitoses were frequent (ca. 1/4 of all spermatogonial mitoses examined) in both the untreated and treated groups. Most of these were considered to have been derived from normal spermatogonia. In busulfan-treated mice, polyploid-like mitoses with PCC were seen. The frequency of PCC-containing mitoses increased with increasing dose and exposure time. A possible interpretation for PCC induction in mouse spermatogonia is discussed.