Regenerative Cell Proliferation Research Articles

Relationship between Hepatocyte Necrosis, Proliferation, and Initiation Induced by Diethylnitrosamine in the Male F344 Rat

Diethylnitrosamine (DEN) is commonly used as an initiator in rodent models of multistage carcinogenesis. Because the initiating activity of DEN has been attributed, in part, to its induction of regenerative cell proliferation, the temporal and quantitative relationships among necrosis, replication, and initiation were characterized in livers of male F344 rats subsequent to administration of a single dose of 10 or 150 mg DEN/kg. Following a dose of 150 mg DEN/kg body weight, maximal hepatocellular necrosis was observed 2 days postinjection and amounted to 9% of the hepatic volume being necrotic by light microscopic criteria. Changes in serum levels of alanine and aspartate aminotransferases, indicators of hepatocellular necrosis, paralleled changes in the necrotic volume fraction. Hepatocyte replication was estimated using nuclear labeling with bromodeoxyuridine (BrdU), which was constantly infused for 2 or 7 days by osmotic minipump. BrdU labeling was maximally increased at 4 days with 2-day infusion (26.1% in treated vs 0.5% in controls) and at 7 days with 7-day infusion (46% in treated vs 2% in controls). Initiation was quantitated by enumeration of hepatocytes which stained positive for placental glutathione-S-transferase (GST-P). Increased numbers of GST-P-positive hepatocytes were observed on Day 4 and increased to a maximum of 109/cm2 section area, or 0.077% of all hepatocytes. Thus, the temporal pattern changes following 150 mg DEN/kg body wt are consistent with the attribution of regenerative cell proliferation contributing to the yield of initiated cells. A comparison of the peak BrdU (2-day) labeling index and the peak GST-P staining frequency suggests a rate of initiation of roughly 10−3-10−4/cell division following 150mg DEN/kg body wt. In contrast, 10 mg/kg DEN did not cause necrosis at Day 2 or increase in BrdU labeling (7-day infusion, 3.1%); however, GST-P hepatocytes were slightly increased over control (1.7 vs 0.0 and 4.1 vs 0.3/cm2 at 14 and 28 days, respectively). Thus, cell replication and yield of initiated (GST-P-positive) hepatocytes in rats receiving 10 mg DEN/kg body wt were both approximately 100-fold lower than those in rats receiving 150 mg DEN/kg body wt. These results indicate the dependence of the frequency of GST-P-positive cells on hepatocyte replication in initiation by DEN and provide crucial response data for quantitative modeling of experimental hepatocarcinogenesis.

Lack of synergism among DMAB, BOP, and MNU in induction of carcinomas of the rat ventral prostate.

The present experiment was undertaken to investigate possible synergism in induction of rat prostate tumor. F344 rats were repeatedly administered the prostate carcinogens 3,2'-dimethyl-4-aminobiphenyl (DMAB),N-nitrosobis(2-oxopropyl)amine(BOP), and N-methylnitrosourea (MNU) sequentially or together at times of hormonal castration induced regenerative cell proliferation of prostate epithelial cells. Group 1 animals received two 100 mg/kg doses of DMAB, two 20 mg/kg applications of BOP, and two times 15 mg/kg of MNU. Groups 2, 3, and 4, respectively, received each of the carcinogen treatment alone. Group 5 was given 6 applications of all three in combination, each at one-third the dose administered to the other groups. The results showed a clear synergism in enhanced tumor development in the colon but not in the prostate, in which the incidence of lesions induced by the three carcinogens was similar to that with DMAB alone.

Early Cell Kinetic Effects of a Single Dose of Monochromatic Ultraviolet B Irradiation on Hairless Mouse Epidermis

Hairless mice were exposed to a single erythemic (25 mJ/cm2) or suberythemic dose (12.5 mJ/cm2) of ultraviolet B (UVB) irradiation at 297 nm. The cell kinetic changes were observed at several times during the first 7 d after the irradiation. The mitotic count, the mitotic rate (stathmokinetic method), and the number of suprabasal and basal cells were scored in histologic sections. The incorporation of [3H]thymidine was measured after pulse labeling, and the DNA distribution pattern was studied by flow cytometry. Initially, both UVB-doses induced a block or delay in the cell proliferation. The rate of entrance of cells into mitosis and the uptake of [3H]thymidine were reduced, and cells accumulated in the S phase of the cell cycle. Hence, during the first period after irradiation, UVB seemed to interfere with the DNA synthesis by inducing a prolonged S phase duration. The DNA synthesis rate was reduced to the same degree after both UVB-doses. From 24 h after irradiation rapid regenerative proliferation took place, most pronounced after the highest UVB-dose. Waves of proliferation seemed to arise from partially synchronized cohorts of cells proceeding through the cell cycle at a higher speed than normal. Thus, the present study indicates that UVB irradiation is comparable with the cell kinetic effects following both chemical skin carcinogens and non-carcinogenic skin irritants. UVB induces an inhibitory effect on the DNA synthesis activity, in addition to regenerative cell proliferation subsequent to cell toxicity.

Distribution of S-phase cells during the regeneration of Drosophila imaginal wing discs

We investigated the distribution of S-phase cells during regeneration of the imaginal wing disc of Drosophila melanogaster following excision of 30°, 90°, and 150° sectors of tissue. The fragments were cultured in adult abdomens for 1–5 days, labeled in vitro with tritiated thymidine, serially sectioned, and subjected to autoradiography. There was negligible thymidine incorporation in unoperated controls and in the undamaged parts of the operated discs, indicating that DNA synthesis in undamaged tissue is terminated during the first day of the culture period. Almost all of the fragments from which tissue had been removed, as well as controls which were simply cut without the removal of any tissue, showed a cluster of labeled cells (blastema) even after only 1 day of culture. The blastemas in control discs were short-lived, with over 50% of these discs showing no blastema by the third day in culture. Blastemas in discs from which sectors were removed were more persistent; the time at which 50% of the fragments no longer showed a blastema was 4 days for the −30° fragments, 5 days for the −90° fragments, and greater than 5 days for the −150° fragments. The average blastema size, measured as number of labeled cells, was directly related to the amount of tissue removed, and in most cases did not change significantly during the culture period. Both wound edges incorporated tritiated thymidine initially and the S-phase cells remained tightly clustered throughout regeneration; maximum blastema width varied from about 8 to 25 cell diameters. The results are consistent with the idea that regenerative cell proliferation is stimulated and maintained by positional information discontinuities, and terminated when these discontinuities are resolved by the addition of an appropriate number of new cells.

Failure of mitogen-induced cell proliferation to achieve initiation of rat liver carcinogenesis.

Experiments were designed to determine whether mitogen-induced cell proliferation is as effective as regenerative cell proliferation in achieving initiation of liver carcinogenesis. To test this hypothesis male Wistar rats were injected with a single dose of diethylnitrosamine (DENA) or N-methyl-N-nitrosourea (MNU) during the peak of DNA synthesis following the administration of the liver mitogen, lead nitrate, after partial hepatectomy (PH) or a necrogenic dose of CCl4. The initiated hepatocytes were monitored as gamma-glutamyltransferase (GGT)-positive foci using a 2-week selection regimen consisting of 0.03% 2-acetylaminofluorene (2-AAF) coupled with a necrogenic dose of CCl4. The results indicate that unlike compensatory cell proliferation such as that induced by PH or CCl4, mitogen-induced cell proliferation did not result in any initiated hepatocytes despite the fact that in both types of models the extent of liver cell proliferation at the time of the administration of the carcinogen is similar.

Proliferationskinetik regenerierender Urothelzellen in der Rattenharnblase nach Applikation von Cyclophosphamid

Regenerative cell proliferation of the urinary bladder urothelium has been investigated in 303 female Wistar rats after damage by single or multiple applications of cyclophosphamide. Between 2 h and 14 h after administering 100 mg/kg, the3H-TdR-index of the entire urothelium was clearly lesser than the control value. After 22 h the proliferative activity increased rapidly and after 30 h a peak value with a mean3H-TdR-index of 17.8% was obtained. Thereafter the labeling index fell to an average value of 4.1% at 2 days. After 3 days there was a renewed rise in the3H-TdRindex, which reached a second peak of 12.2% on the 7th day. After application of 200 mg/kg the3H-TdR-index increased in a similar manner at 22 h, following an initial depression of DNA synthesis. After 32 h the highest labeling index of 12.2% was obtained. At about 4 weeks both doses of cyclophosphamide yielded values corresponding with those in the control animals. The3H-TdR-index rose initially in the trigone region and also reached its highest value there. After about 1 week the maximum proliferative activity had shifted upwards into the dome of the bladder. The basal cells showed a considerably higher absolute labeling index than the suprabasal cells. After multiple intermittent applications a slightly increased3H-TdR-index was found with the increase in the total dose administered. Using the percentlabeled-mitoses method, average values were obtained of 24 h for the generation time (TC), 15 h for the DNA-synthesis phase (TS), 7 h for the postsynthetic phase together with the mitotic phase (TG2 + M) and 2 h for the presynthetic phase (TG1). Stimulation of the proliferative activity of rat bladder urothelium by cyclophosphamide provides itself as an experimental animal model for testing weak carcinogenic agents.

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.