Interaction of chalones and glucocorticoid hormones in regulation of cell division

The action of hepatic chalone on cell proliferation in inoculated hepatoma 22a of mice was studied in the presence of a changed level of glucocorticoid hormones in experimental animals. Chalone was obtained from the liver of intact rats by ethanol precipitation. The intensity of cell proliferation in hepatoma was evaluated by the colcemide and autoradiography methods. Six hours after chalone injection c-mitosis in the tumor decreased 2.7-fold, and the DNA index 6.8-fold. It may be concluded that the preparation used contains both G1- and G2-chalones. Single or repeated injections of hydrocortisone to mice inhibits cell proliferation to a less degree than administration of chalone alone. Combination of hydrocortisone and chalone produces the same effect as injection of chalone alone. Adrenalectomy diminishes susceptibility of hepatoma cells to exogenous chalone. The degree of tumor proliferative activity in the adrenalectomized animals was half as much after chalone injection, as compared to that in intact animals. Thus, a certain level of glucocorticoid hormones in hepatoma tissue is necessary to reveal the action of chalones.     

Effect of tissue-specific proliferation inhibitors on the level of mitotic activity and cell adhesion in disordered innervation

In the work there was studied the influence of hepatic chalones on the level of mitotic activity and on the degree of adhesion of hepatocytes after the violation of parasympathetic and sympathetic innervation under the physiological conditions of liver function and in regenerating organ. Some definite regularities were revealed in the change of the power of linkage among the cells of liver parenchyma after the disturbance of its innervation and chalones affected. Significant differences in the effect of the influence of tissue inhibitors of proliferation on the process of regeneration in liver, which has intact or disturbed innervation were discovered. Particularly one can underline the effect of the loss of hepatocytes sensitivity to chalones in vagotomized liver.     

G1 and G2 chalones of the gastric mucosa

A study was made of the action of human gastric mucosa G1 and G2 chalones on cellular regeneration of mouse gastric mucosa and of the duration of their maximal effect. Chalone fractions were obtained from the mucous membranes of 21 stomachs resected for peptic ulcer by the method of fractional ethanol precipitation. The data indicate that the maximal inhibitory action of G1 chalone occurs in 3, whereas that of chalone G2 in 6 hours. Some specificity of the action of chalones was discovered depending on the part of the gastric mucosa from which they were obtained.     

A quantitative model of liver regeneration in the rat

The ability of various theories to generate the kinetics of rat-liver regeneration is considered. Wound hormone and functional demand theories are shown to be either inadequate or overly complex. A simple model based on a liver-produced mitotic inhibitor is, however, able to match the experimental results on the mitotic rate and thymidine uptake of parenchymal cells in the liver as a function of time following various degrees of partial hepatectomy. The inhibitor itself would belong to the class of molecules known as chalones and differential equations describing the mechanism of its action are derived and solved numerically. The only arbitrary parameters required for the solution are those giving the halflife and dose-response curve of the inhibitor. Optimal matching of theory to data is obtained when the half-life is about 3 h and the dose-response curve is given by a negative exponential function. Experimental procedures for measuring these parameters are discussed and an explanation of the uneven distribution of mitoses in regenerating liver is given.     

Effect of epidermal chalones on the development and growth of cheek pouch tumors in the hamster

To the cheek pouch mucosa of 118 male Syrian golden hamsters 0.5% acetone solution of 7,12-dimethylbenz (a) anthracene was applied 3 times a week during 2 months. Two months after beginning of the experiments all the hamsters developed tumors. Since that time a mixture of epidermal G1 and G2 chalones was applied to the cheek pouch mucosa of the experimental animals 5 times a week. The control hamsters received saline alone. As compared to the control, the experimental hamsters demonstrated an increase in the life-span, tumor growth retardation and regression of part of the tumors. The data obtained attest to the anti-neoplastic action of chalones.     

ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve

Adrenocorticotropin (ACTH)-treated adrenalectomized rats subjected to crush denervation recover sensation and functional movement sooner than saline-treated rats. Axonal regeneration is accelerated, the number of large endplates and the frequency of preterminal branching are increased. ACTH has no effect on either intact or denervated muscles. The ameliorative action of ACTH during regeneration is apparently neurogenic and independent of corticoids.     

Course of liver regeneration after partial hepatectomy in rats treated with dialysates of intact organs

A number of growth phenomena observed in vitro have shown that cells, at high densities, produce and release substances which, when they have reached a given concentration, arrest further growth. In vivo, these possibilities can be studied on the model of rapid regeneration of the rat liver after 65-70% partial hepatectomy (PH). We evaluated the course of liver regeneration after PH in animals treated with dialysates (DIA) of intact rat tissues. In addition to kidney and lymph node DIA, we were particularly interested in the effect of liver and spleen DIA. The experiments were carried out on male rats weighing 210-240 g. The relevant DIA was administered 24 h prior to PH; the controls were given physiological saline. The animals were killed just before PH and 24, 48, 30 and 72 h and 14 days after. DIA obtained from intact liver tissue inhibited the regeneration process induced by PH and its effect persisted 48 h after PH. Compared with the controls and with the rats given kidney DIA, DNA synthesis in the liver 24 h after PH was reduced to 77%. After spleen DIA, several (still hypothetical) factors probably acted together synergically (factors belonging to the immune system--RES--and spleen-produced factors capable of promoting proliferation of the hepatocytes--the "portal blood factor"). We arrived at this conclusion from an evaluation of liver DNA synthesis 24 h after 24h after PH, when synthesis was altogether markedly raised, but attained far higher values after the administration of spleen DIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pregnenolone-16alpha-carbonitrile, a microsomal enzyme inducer, on the regenerating rat liver.

PCN, a microsomal enzyme inducer, given orally (10 mg in 1 ml water twice daily for 5 days), increased liver weight and mitotic activity in intact as well as in partially hepatectomized rats. Electron microscopy revealed SER proliferation in the hepatocytes of animals treated with PCN alone. Accumulation of SER membranes was also evident in the liver cell cytoplasm of untreated, partially hepatectomized rats; it was however, more pronounced in the hepatocytes of partially hepatectomized animals given PCN. These results indicate that the steriod has a marked effect on the regeneration rat liver.     

Levels of cyclic GMP and cyclic AMP in regenerating forelimbs of adult newts following denervation

The effect of short-term denervation (0, 12, 24, and 72 hours) on the levels of cyclic 3'5'-guanosine monophosphate (cGMP) and cyclic 3'5'-adenosine monophosphate (cAMP) in adult newt (Notophthalmus viridescens) forelimbs at 15, 22, and 35 days of regeneration was investigated. Regenerate blastema and stump cyclic nucleotide levels were compared with those of the contralateral intact forelimb and hindlimb, with levels in the normally regenerating blastema, and with levels measured in the forelimbs of intact, nonoperated animals. Variations in cyclic nucleotide levels occurred according to regeneration stage and tissue type. Changes in level were noted immediately upon denervation and subsequently at other sample times in all regenerate and control series. Parallel fluctuations occurred in regenerate stump and contralateral intact forelimbs. Our results from nonamputated denervated and sham-denervated animals indicate that short-term, denervation-associated cyclic nucleotide fluctuations cannot be attributed solely to the loss of innervation.     

Effect of carnosine and 4-methyluracil on the development of experimental hepatitis in rats]

A comparative study of the hepatoprotective effect of carnosine and 4-methyluracil under CCl4-induced acute toxic hepatitis has been carried out. The extent of liver injury and its regeneration were established from morphological data as well as from changes in the activities of alanine aminotransferase (ALT) and histidase and the bilirubin content in blood serum. Hyperlipoperoxidation in the liver and serum was assessed by the amount of TBA-active products. It was found that by day 10 of experimental hepatitis ALT and histidase levels in blood sera of untreated animals exceeded the normal values 1.3- and 3.9-fold, whereas those in the carnosine-treated group approximated the values characteristic of intact animals. The activity of serum ALT in animals treated with vitamin B12 or 4-methyluracil exceeded normal values 1.5 and 1.6 times, whereas that of histidase was 2.5 and 2.7 times as high. Carnosine and 4-methyluracil inhibited (in approximately the same degree) the formation of TBA-active products in the liver. According to morphological dta, cessation of CCl4 injections was accompanied by rapid regeneration of liver tissues in all animal groups. Carnosine enhanced regenerative processes in parenchymatous and connective tissues in a far greater degree in comparison with other drugs. The mitotic index in the carnosine-treated group exceeded more than twofold the corresponding parameters in untreated animals. Possible mechanisms of carnosine action on liver repair are discussed.     

VARIATIONS OF MITOSIS-INHIBITING CHALONE ACTIVITY IN EPIDERMIS AND DERMIS AFTER CARCINOGEN TREATMENT

During regeneration and hyperplasia following a single application of a carcinogen to mouse epidermis, the rate of epidermal cell proliferation varied inversely with the content of mitosis-inhibiting activity (chalone). The main inhibitory activity was present within the epidermal cells, but a lesser activity was also found in the corresponding extracts of dermis. This supports the concept that chalones act as physiological regulators of cellular proliferation under pathological as well as normal conditions. The possibility that the dermis also takes an active part in this regulation is discussed.     

Cellular mechanisms of cirrhotic rat liver regeneration. II. Proliferation, polyploidization and hypertrophy after partial hepatectomy

Using cytofluorimetry and absorptional cytophotometry, hepatocyte DNA and total protein contents were measured in intact and cirrhotic rats in 1, 3 and 6 months after partial hepatectomy (PH). It has been found that within one month of intact rat liver regeneration the level of hepatocyte ploidy rised by 25% to remain elevated for the next 6 months. This was due mainly to reducing the number of cells with diploid nuclei (2c 2-fold, 2c x 2 - 6.6-fold) and to rising the number of octaploid hepatocytes. In cirrhotic animals the ploidy level in hepatocytes increased in 3 months after PH, and decreased by 15% in 6 months. The number of hepatocytes with diploid nuclei (2c and 2c x 2) increased within 3-6 months in both control and cirrhotic rats. The protein content per diploid hepatocyte rised by 30% within 3-6 months of liver regeneration after PH. Special calculations have shown that within 3 months after PH the increase in the liver mass of control and cirrhotic rats was due completely to hepatocyte DNA synthesis, i. e. proliferation and polyploidization. Within the next 3 months of liver regeneration after PH, the contribution of polyploidization to liver mass increase was negative because of depolyploidization of liver parenchyma cell population. At this time hypertrophy was the main process determining the liver mass increase.     

DNA synthesis in rat liver following administration of antihepatocytotoxic serum]

Experiments were conducted on adult female rats. The autoradiographic method was applied to the study of thymidine-3H incorporation into the parenchymatous and reticulo-endothelial cells of the liver under conditions of using low doses (0.06 microgram of protein per 100 of body weight) of antihepatocytotoxic serum (AHCS), gamma-globulin isolated from it (gammaAHCS) and gamma-globulin fraction of normal rabbit serum (gammaNRS) to intact animals and rats with carbon tetrachloride affection of the liver. The labelled nuclei index of both the parenchyma and the reticuloendothelial cells increased in case of gammaAHCS administration, and, to a lesser extent, of AHCS to intact animals. gammaAHCS used against the background of CCl4 administration intensified the reparative regeneration. The action of gammaAHCS has phasic character--the period of the labeled nuclei elevation was followed by their reduction, replaced by new intensification of the proliferative processes.     

Local regeneration in the retina of the goldfish

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transsclearal surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2′-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithiallike cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

Local regeneration in the retina of the goldfish.

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transscleral surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2'-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithial-like cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

Activity of enzymes of nucleic acid metabolism in rat liver nuclei under induction of RNA synthesis by electric stimulation of hypothalamus]

Electrostimulation of hypothalamus results in an increase of RNA synthesis of the AU-type in liver cells of intact and adrenalectomized rats. No such effect is observed in the animals with denervated liver. An increase in RNA synthesis is accompanied by an increase in the activities of RNA-polymerases II and III and histone hydrolase and a decrease in the activities of RNAse and DNAse. The mechanisms of the effect of the nervous system on the activities of the nucleic acid metabolism enzymes are discussed.     

Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts

Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with ³H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.     

Molecular dissection of gp130-dependent pathways in hepatocytes during liver regeneration

Interleukin-6 (IL-6) via its signal transducer gp130 is an important mediator of liver regeneration involved in protecting from lipopolysaccharide (LPS)-induced liver injury after partial hepatectomy (PH). Here we generated mice either defective (Delta) in hepatocyte-specific gp130-dependent Ras or STAT activation to define their role during liver regeneration. Deletion of gp130-dependent signaling had major impact on acute phase gene (APG) regulation after PH. APG expression was blocked in gp130-DeltaSTAT animals, whereas gp130-DeltaRas mice showed an enhanced APG response and stronger SOCS3 regulation correlating with delayed hepatocyte proliferation. To define the role of SOCS3 during hepatocyte proliferation, primary hepatocytes were co-stimulated with IL-6 and hepatocyte growth factor. Higher SOCS3 expression in gp130-DeltaRas hepatocytes correlated with delayed hepatocyte proliferation. Next, we tested the impact of LPS, mimicking bacterial infection, on liver regeneration. LPS and PH induced SOCS3 and APG in all animal strains and delayed cell cycle progression. Additionally, IL-6/gp130-dependent STAT3 activation in hepatocytes was essential in mediating protection and thus required for maximal proliferation. Unexpectedly, oncostatin M was most strongly induced in gp130-DeltaSTAT animals after PH/LPS-induced stress and was associated with hepatocyte proliferation in this strain. In summary, gp130-dependent STAT3 activation and concomitant SOCS3 during liver regeneration is involved in timing of DNA synthesis and protects hepatocyte proliferation during stress conditions.     

Neurite outgrowth and proliferation of non-neuronal cells on cryostat sections of adult muscle

Rat spinal cord cells were cultured on cryostat sections of innervated and denervated muscle. Neurite outgrowth was greater on sections of denervated muscle, which therefore appeared to act on in vivo nerve regeneration. It seems that muscle sections were able to release into the culture medium factors that increase proliferation of fibroblasts. The muscle therefore appeared able to modulate its interaction with its environment by acting on different types of cells.