Increase In Proliferative Activity Research Articles

Early mucosal changes in dimethylhydrazine-induced colonic carcinogenesis in rats.

Early mucosal changes in dimethylhydrazine (DMH)-induced colonic carcinogenesis were investigated in 12 rats. These rats received subcutaneous injections of DMH in a dose of 15 mg/kg/week. Two rats each were killed at 6, 8, 10, 14, 18 and 22 weeks after initiation of the DMH-treatment. Overt carcinomas developed in 3 out of 4 rats killed after the 18th week of DMH-treatment. Histopathologically, epithelial dysplasia was present in the flat colonic mucosa of all 6 rats killed after the 14th week of DMH-treatment. Neither grossly visible tumors nor histopathological dysplasia was present in the colon of the 6 rats killed before the 14th week of DMH-treatment. However, cytophotometric DNA analysis disclosed that significant increases in proliferative activity of mucosa had occurred 4 weeks before the appearance of histopathological dysplasia, and 8 weeks prior to development of grossly visible tumors. Therefore, changes in the flat mucosa of a tumor-bearing colon represents a primary precancerous condition.

16,16-Dimethyl prostaglandin E2 suppresses the increases in the proliferative activity of rat colonic epithelium induced by indomethacin and aspirin

Treatment of rats with indomethacin rapidly increased ornithine decarboxylase (4 h) of colonic mucosa and [3H]thymidine incorporation into coJonic mucosal deoxyribonucleic acid (DNA) (1 or 5 days) when this parameter was examined in vivo and ex vivo. The changes in colonic mucosal ornithine decarboxylase and DNA synthesis induced by indomethacin were correlated temporally with suppression of colonic prostaglandin synthesis, as assessed from ex vivo colonic production of prostaglandin E, the dominant prostaglandin product of colon. Autoradiographic studies indicated that the enhancement of proliferative activity of colonic epithelium after treatment with indomethacin for 1 day was confined to the lower third of the colonic crypt (normal proliferative zone). After 5 days of indomethacin treatment, however, there was an extension of the proliferative zone to the upper third of the colonic crypts. Concurrent treatment of rats with the stable prostaglandin E2 analogue, 16,16-dimethyl prostaglandin E2, suppressed indomethacin-induced increases in colonic mucosal ornithine decarboxylase and DNA synthesis. Concurrent administration of 16,16-dimethyl prostaglandin E2 also prevented the extension of the proliferative zone of colonic epithelium induced by 5 days of indomethacin administration. 16,16-Dimethyl prostaglandin E2 alone for 1–5 days had no detectable effects on colonic mucosal ornithine decarboxylase and DNA synthesis compared with corresponding control values. Increases in colonic mucosal DNA synthesis were also induced by treatment of rats for 5 days with aspirin (ASA). The stimulation of colonic mucosal DNA synthesis induced by ASA was significantly suppressed by concurrent administration of 16,16-dimethyl prostaglandin E2 and was also correlated with the inhibition of colonic prostaglandin synthesis by ASA. The colons of rats treated with indomethacin for 1 day or ASA for 5 days appeared normal by light microscopy. However, treatment of rats for 5 days with indomethacin resulted in mild to moderate inflammation of the lamina propria and some goblet cell depletion at the mucosal surface, but no loss of surface epithelium. The ultrastructure of the surface epithelium of the colons of rats treated with indomethacin or ASA was normal as assessed by electron microscopy. The results thus demonstrate that inhibition of local colonic prostaglandin synthesis is associated with increases in the proliferative activity of colonic epithelium, and that these increases are suppressed by administration of 16,16-dimethyl prostaglandin E2. Under some conditions, the increases in proliferative activity that were observed with indomethacin and ASA are dissociated from drug-induced inflammation and cell surface injury.

Digestive tract cell proliferation and food consumption patterns of Ha/ICR mice.

The relationship between the daily pattern of food consumption and the proliferation rate of the oesophagus, stomach, forestomach, small intestine and colon of Ha/ICR mice was examined. Proliferative activity was determined by [3H]TdR incorporation on a wet weight tissue basis, along with selective counting of labelled nuclei. Under conditions of ad libitum feeding with a 12 hr light cycle (lights on at 0600) mice eat most of their food during the dark period. A distinct circadian rhythm was observed in the oesophagus, stomach, forestomach and colon with the peak of [3H]TdR incorporation between 0400 and 0600 and the nadir between 1600 and 1800. Although a circadian fluctuation was observed in the small intestine, its amplitude was much less than in other areas. This rhythmic change in proliferation rate could be phase shifted by allowing the mice to feed only between 0800 and 1600 for 14 days. Under these conditions the peak in proliferative activity occurred between 1800 and 2000. Fasting reduced the daily level of proliferative activity in all of the digestive tract sites studied, and for all areas except the oesophagus greatly reduced or eliminated the circadian fluctuation. The forestomach and colon were the most influenced by fasting with 24 hr [3H]TdR incorporation reduced to 30-40% of the control value. Refeeding following a 48 hr fast produced a rapid increase in proliferative activity peaking at levels well above the control value at 16 hr after the onset of refeeding. The major exception to this was the small intestine which slowly returned to the control value during the first 24 hr. Partial refeeding produced a diminished refeeding response. Once the normal pattern of food consumption was re-established following refeeding the normal proliferative fluctuations were again observed.

Cell kinetics in normal and neoplastic mammary tissues by flow cytometric analyses.

The cell cycle kinetics of normal mammary tissue and cancer mammary tissues were studied by flow cytometry to determine the effect of estrogen and progesterone and antiestrogens on proliferative activity. The proliferative activity was higher in rat mammary tissue during the estrus and pregnant stages compared to the nonestrus or lactational stages. Upon 5 days administration of estrogen and progesterone to the lactating animal, the low proliferative activity was increased to the values found in the pregnant stage. Steroidal effect on proliferative activity was also shown using organ cultures of normal rat tissues and the increase in proliferative activity can be reversed by the addition of 10(-6) M retinoic acid but not by retinyl acetate. In a human mammary tumor cell line (MCF-7), the cell kinetic activity was significantly depressed by antiestrogens (i.e., tamoxifen) as determined by both flow cytometry and tritiated thymidine uptake studies. Finally, a human tumor model system was developed with the MCF-7 cell line in estrogenized nude mice. Flow cytometry of the resulting tumor indicates a tetraploid tumor similar to the cell culture. Cell kinetic analysis of these nude mice tumor cells demonstrates the high proliferative activity of the estrogenized animals compared to the tamoxifen-treated animals. In conclusion, the use of flow cytometry to rapidly measure cell kinetic activity of mammary tissue has been demonstrated. The effects of estrogen or estrogen and progesterone hormones seem to increase the proliferative activity, while antiestrogens decrease this activity. Findings are observed in both in vivo and in vitro conditions or normal tissues, mammary cancer tissue, and cultured cells.

Interaction of inhibitor and stimulator in the regulation of CFU-S proliferation

An inhibitor and stimulator of CFU-S proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-S (e.g. normal bone marrow) and proliferating CFU-S (e.g. regenerating bone marrow). In this paper we explore the capacity for inhibitor and stimulator production in relation to changes in the proliferative status of the CFU-S and their mode of interaction. The increase in proliferative activity of CFU-S following a concentrated regime of phenylhydrazine injections is paralleled by an increase in the capacity for stimulator production and a decrease in the capacity for inhibitor production. These processes are reversed as the normal low proliferative activity returns. Using density fractionated marrow populations, dose-response measurements show that while inhibitor- and stimulator-producing cells persist throughout the changes in CFU-S proliferation, their magnitude of factor production moderates in relation to those changes. The inhibitor and stimulator are not mutually destructive: the two factors retain their activities after co-incubation and re-separation. On the other hand, the presence of either factor blocks the synthesis of the other by the appropriate producer cells. A model for the regulation of CFU-S proliferation by the inhibitor and stimulator is thus suggested in which the relative spatial distributions of CFU-S in a microenvironment containing inhibitor- and stimulator-producing cells are an important feature. It also implies the existence of a signal from the CFU-S compartment which determines the appropriate inhibitor or stimulator production.

Biophysical Characterization of Normal T-Lymphocytes and Sézary Cells

Blood lymphocytes from 18 patients with cutaneous T-cell lymphoma (Sézary syndrome and mycosis fungoides) were characterized using multiparameter laser flow microfluorimetry (FMF) and automated image analysis (AIA) and the results correlated with routine blood smears, cytogenetic studies and observations made on PHA-stimulated normal T-lymphocytes in vitro. Specimens from all 9 patients with Sézary syndrome and 5 of 9 patients with mycosis fungoides contained one or more discrete subpopulations of neoplastic (Sézary) lymphocytes that were detected by FMF. Studies with AIA demonstrated that neoplastic T-lymphocytes are distinguished from normal quiescent (G0) lymphocytes not only by alterations in DNA content (aneuploidy) but also by chromatin structuring (increased chromatin dispersion), which may be a more sensitive index of neoplastic transformation than ploidy levels. In several patients, small and large Sézary cells were present with DNA-chromatin properties quite similar to normal cycling G1 and G2 lymphocytes respectively, but their presence was not explained by an increase in proliferative activity in the blood. These findings indicate that Sézary syndrome consists of a heterogeneous group of related disorders differing in terms of the Sézary cell population. The response to treatment and prognosis may differ accordingly.

Proliferation kinetics of the mouse bladder after irradiation.

The proliferation response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose-dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing this response was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.

Epithelial cell kinetics in the descending colon of the rat. I. The effect of ischaemia-induced epithelial cell loss.

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.

Diurnal rhythm of changes in the number of DNA-synthesizing and mitotically-dividing jejunal epithelial cells after subdiaphragmatic vagotomy

Bilateral subdiaphragmatic vagotomy in rats led to an increase in proliferative activity of the jejunal epithelium 7 days after the operation without any accompanying change in the diurnal rhythm of cell division. Meanwhile, after vagotomy the phase structure of the rhythm of DNA synthesis was modified in the intestinal epithelial cells: from the normal unimodal type it became bimodal.

Clinical aspects of alpha 1-fetoprotein determination in human liver cancer and in humans and experimental animals at carcinogenic risk.

Recent findings concerning the significance of alpha 1-fetoprotein (AFP) as a tool for clinical diagnosis and monitoring of tumor diseases are reviewed briefly. The applicability of this protein marker to the early diagnosis of patients at carcinogenic risk is discussed. In addition, experimental data obtained with a model of chemical hepatocarcinogenesis are reported. The increase of proliferative activity in precancerous liver tissue preceded AFP production under experimental conditions with azodyes and aflatoxin B1 as carcinogens. Immunohistochemical analysis of the relation of AFP to changes of cell populations and to liver tissue rearrangement led to the conclusion that AFP-producing cells cannot be precursors of malignant hepatocytes; however, AFP appeared to be linked to dividing hepatocytes at a certain step of cell differentiation regardless of the stages of precancerous development. A decrease in the rate of nuclear RNA synthesis was observed in both precancerous and tumor tissues. A possible analogy between the early phase of AFP production in animal carcinogenesis and that in human carcinogenesis is considered.

The role of hyperlipidemic serum on the proliferation and necrosis of aortic medial cells in vitro

The effects of hyperlipidemic rabbit serum on the proliferation and death of subcultured rabbit aortic medial cells were evaluated. The cultured cells were grown from the aortic media of normal adult male rabbits, prelabeled with [ 3H]thymidine, and then trypsinized, pooled, and subcultured. Either normal or hyperlipidemic rabbit serum was added to the culture media. Proliferative activity was measured by cell count and by the isotopic dilution of the prelabeled DNA, with corrections made for cell loss. Cell loss, presumed to represent mainly cell death, was assessed from the total loss of [ 3H]thymidine from the cultured cell population. The cells grown in basal medium of Eagle containing 10% normal serum showed a 25% loss in the first 3 days after trypsinization. The loss rate stabilized at 1.5% per day 5 days after the subculture. When these cells were exposed to hyperlipidemic serum, the rate of cell loss showed a 5.5-fold increase in the initial 3 days, and thereafter returned to the levels seen in the presence of normal serum. Cells exposed to hyperlipidemic serum also showed a 25–50% increase in proliferative activity. These alterations have been demonstrated in both the early proliferative phase and later stationary phase. Cells grown in hyperlipidemic serum were larger on the average and showed a 50% increase in protein content per cell.

Increase of Proliferative Activity of Leukaemic Blast Cells from Human Peripheral Blood in Liquid Culture

The proliferative behaviour of leukaemic cells from the peripheral blood of 12 patients suffering from acute leukaemia was investigated in short term liquid culture. In 3 cases of AML an increase of the blast cell number was observed exceeding the initial value whereas in 9 other cases the cell number decreased more or less rapidly. In 9 of these patients the proliferation kinetics of the cultured leukaemic blast cells were studied with 3H-thymidine labelling. In all these cases the labelling index increased during the first days of culture, in 3 cases to values of 48%, 45% and 38% on day 3. Only in 5 cases, however, did an absolute increase of the blast cells incorporating 3H-thymidine occur. Here the doubling times of the proliferating leukaemic blast cells were estimated to be 12, 13, 14, 28 and 55 h. Since a doubling time of 12 to 14 h seems to be too short to be explained only by an exponential growth of the initially proliferating cells it is postulated that leukaemic blast cells in a G0- or long G1-phase were present in the peripheral blood of these patients and that these entered the cell cycle during liquid culture.

MAMMOTROPHIC ACTIVITY IN RAT SERUM DURING THE OESTROUS CYCLE, PREGNANCY AND LACTATION

The mammotrophic activity of various serum samples was assessed on the mammary gland of pregnant rats, in organ culture. Insulin was added to the medium. Serum samples from virgin rats showed little activity, and variation in mammotrophic activity during the oestrous cycle was slight. During pregnancy a significant increase in proliferative activity was not seen during the first week, but around day 8-9 mammotrophic activity increased sharply. The activity showed maxima around days 12 and 19. A decrease in the activity between these maxima was not consistently observed. Mammotrophic activity was still present in sera collected during parturition, and 30 min after parturition. It had disappeared completely within 24 h. The mammotrophic factor detectable by organ culture in the serum of pregnant rats could be rat chorionic mammotrophin. Activity in the serum on day 0 and 1 of lactation was comparable to that of virgin rats, but some activity appeared on day 2. High actvities were foound frequently around day 4 to 6 of lactation. On other days the activity showed fluctuations without a definite pattern. Litter size was of minor importance but the combination of a larger litter size and fasting-overnight seemed to suppress the presence of mammotrophic activity in the serum. Nursing was important: after weaning the activity has disappeared, while renewed nursing after weaning resulted in the appearance of high levels of activity. The mitotic activity obtained with lactating rat serum in the culture was suppressed by addition of rabbit anti-rat prolactin serum to the medium. This suggests that the main mammotrophic factor detectable by organ culture in the serum of lactating rats, is prolactin.

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.

Isolation and purification of mediators of cell proliferation.

An attempt was made to isolate and purify the important biological mediators that cause an increase in proliferative activity of fibroblasts following tissue injury. DNA synthesis and cellular growth, using cultured WI-38 fibroblasts, and DNA synthesis in an in vitro assay, using purified DNA polymerase, were stimulated by factors extracted from the lysosomal-mitochondrial fraction of normal guinea pig liver. These factors precipitated in 45 percent to 60 percent ethanol. They were insensitive to treatment with RNase, DNase and heating to 56 C for 30 minutes, but were inactivated at 100 C. isoelectric focusing of the active ethanol-precipitate resolved activity into five discrete fractions, one of which has been purified, using ion-exchange chromatography. The presence of these factors in normal tissue may explain the increase in proliferative activity of fibroblasts and other cells in the early stages of wound healing, via release caused by injury.

The cell cycle of ventricular cells in the overgrown optic tectum

The cell cycle in the overgrown optic tectum of the chick embryo was investigated radioautographically from the 3rd through the 6th day of incubation. Quantitative data were obtained on the length of the DNA-synthetic period (S), the premitotic period (G 2), mitosis, and the postmitotic period (G 1). At each stage of development, the total generation time in the abnormal brain showed an increase of approximately 2–3 h over that observed in the normal brain. The duration of mitosis, G 1, and the S period likewise increased. The results indicate that although one sees an increased number of mitotic figures during overgrowth, this may reflect a longer period of time spent in mitosis, rather than an actual increase in proliferative activity. This suggests that the term ‘overgrowth’ is a misnomer for a condition which actually involves a slowing down of the proliferative process.

Autoradiographic in vitro-examination of psoriatic skin before, during and after dithranol treatment

Autoradiographic in vitro-examination of normal and of psoriatic skin, using the double labelling technique (1 µC thymidine-2-C14 and 10 µC thymidine-methyl-H3), revealed the usual increase of proliferative activity in psoriatic skin, approximately four times greater than normal. The cell cycle time was shortened. According to the data obtained by the double labelling technique a shortening of the DNA synthesis time was not found in vitro. Treatment with a dithranol-salicylic acid (10%)-petrolatum ointment (DSP) normalized the cell cycle in accordance with the degree of clinical improvement. The findings seem to indicate that DSP has an effect on the DNA-synthesis.

THE PROLIFERATIVE ACTIVITY OF THE ADRENAL CORTEX USING A RADIOAUTOGRAPHIC TECHNIC WITH THYMIDINE-H3.

AbstractProliferative activity of the adrenal cortex was studied following injections of thymidine‐H3. Normal male Long‐Evans rats had less activity than comparable animals of the Sprague‐Dawley strain. Of the Long‐Evans rats killed at different hours of the day, those injected with thymidine‐H3 at 6 P.M. and killed six hours later had the greatest activity. Normal male rats 35 to 73 days of age had almost all activity in the glomerulosa and outer fasciculata of the cortex. This activity was greatly increased two or three days after injecting colchicine, compound 48/80, or carbon tetrachloride. Such stressing agents did not cause any increase in activity during the first 24 hours. Three days after carbon tetrachloride a considerable increase in proliferative activity occurred in the inner zona fasciculata and reticularis of the adrenal of female rats during diestrus. Male rats killed 28 to 40 days after injecting thymidine‐H3 showed no evidence of centripetal migration of cells into the reticularis during that time. The absence of centripetal migration and the marked responsiveness of the inner fasciculata and reticularis of female animals after stressing agents supports the zonal theory of function in the adrenal cortex.

The effect of sex hormones on comb growth in immature pullets.

1. The effects of injecting oestradiol dipropionate (ODP), testosterone propionate (TP) and progesterone (P) in immature pullets have been studied. 2. TP evoked hypertrophy of the comb and wattles, but ODP and P were without effect. 3. The greatest comb growth was observed when all three hormones were injected; next in order were combs from birds receiving TP alone and then those given TP with either ODP or P. 4. Injections of all three hormones produced a slight increase in proliferative activity of the stratum germinativum and a decrease in the density of the connective tissue of the corium of the combs when compared with those of birds injected with TP alone. The combs of birds treated with ODP showed prominent blood capillaries in the peripheral layers of the corium when frozen sections were stained with Scharlach R. This is thought to have been due to the marked affinity of their red blood cells for the stain and indicates a high lipid content of the cell membrane. Similar differences were also noted in the combs of normal laying female as compared with normal male birds. 5. These histological differences provide a possible explanation for the known differences in shape, colour and texture between female and male combs. 6. It is suggested that the histological examination of the comb provides a means of establishing the validity of an assay of a hormonal preparation by the comb growth technique.