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.     

Combined effect of epinephrine and chalone extracted from Ehrlich ascites carcinoma administered during different time of day on cell division in the tumor

The biphasic circadian rhythm of mitotic activity has been demonstrated in a 5-day Ehrlich's ascites carcinoma (EAC) in mice. Adrenaline injected intraperitoneally in a dose of 1.5 micrograms/g bw produced an inhibitory effect on cell division that lasted over 4 hours and reached maximum at injection to mice during light time of the day. EAC extract in a dose of 1 ml also inhibited the mitosis during 4 hours, but the greatest fall in the mitotic activity was observed during the minimum mitotic activity in the control animals. Combined administration of adrenaline and the extract resulted in the phenomenon of prolonged inhibition of cell division, that persisted for maximum 6-8 hours, if the preparations were injected in the middle of the day light time. Of definite importance was the rhythm of changes in the sensitivity of proliferating tumor cells.     

Effect of a chalone-containing preparation from Ehrlich ascites tumor on DNA synthesis and cell division in the tumor in relation to the time of day

The mitosis inhibitory action of chalone-containing preparation of the Ehrlich ascite tumour was shown to depend on the time of its administration on round the clock, and on the circadian rhythm phase of the mitotic activity in this tumour. This allowed a conclusion that the chalone system of the tumour may be involved in the formation of the circadian rhythm of cell division. It was found that Ehrlich's ascite tumour chalone system regulated DNA synthesis influencing the cell passage from G1-phase of the mitotic cycle to S-phase, and the processes occurring during S-phase.     

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.     

Action of a chalone-containing esophageal preparation on its epithelial cell proliferation

The effect of esophageal chalone on epithelial cell reproduction in the esophagus was studied. Lyophilized aqueous extract from the esophagus was used. The following properties of the esophageal preparation have been revealed: it is water-soluble; it is present in the same tissue where it acts; it has tissue-specific effect (the preparation does not act on the mitotic and radioactive index in the epithelial crypts of the small intestine); its action is short-term and reversible; its effect on DNA division and synthesis in the esophageal epithelial cells is dose-dependent. Therefore, it is suggested that the esophageal preparation contains a chalone.     

Relation between the mitosis-inhibitory activity of a chalone preparation of Ehrlich ascites tumor and its diurnal secretion

Mitosis inhibitory activity of chalone-containing preparations extracted from cells of Ehrlich's ascites carcinoma (EAC) at varying time of day (9, 13, 17, 21 1, 5 and 9 o'clock) was studied in a temporary suspension culture of EAC. The inhibitory action of the preparations depended on the time of their extraction. This may be indicative of rhythmical alterations of the production or activity of EAC chalone. The maximum activity was demonstrated by the preparations extracted at 5, 9 and 13 o'clock. The activity of the preparations, extracted at 17, 21 and 1 o'clock was considerably less. The degree of mitotic activity inhibition also depends on the dose of duration of action of the chalone-containing preparation.     

Chalones and the Control of Normal, Regenerative, and Neoplastic Growth of the Skin

SYNOPSIS Chalones,inhibitors of cell dmsion have been isolatedand studied from a number of mammalian tissues, most notably,the epidermis The epidermal rhalone is a glycoprotein It exhibitsconsiderable, but not complete specificity The epidermal chalone decreases mitotic activity by inhibitingcells in the G 2 phase of the cell cycle from entering mitosis,and probably also by inhibiting ceils in the G 1 phase of thecell cycle from entering mitosis To inhibit cells in G 2 fromentering mitosis the chilone requnes adrenalin, and for maximalactivity hydrocortisone It is not known if idrenalin and hydrocortisoneare required for chalone inhibition of cells in G 1 In addition to inhibiting cell division in normal epidermalcells the epidermal chalone can inhibit cell division in regeneratingepidermal cells induced to proliferate by chemical damage Thephase of the cell cycle in which the chalone inhibits legeneratingepidermal cells from entering mitosis is not known Epidermal tumors contain a decreased amount of chalone Mitosisin epidermal tumors is inhibited by treatment with epidermalchalone Tumor cells are inhibitedfrom entering mitosis fromeither the G 1 or G 2 phases of the cell cycle Chalones are said to inhibit mitosis by a negative feedbackmechanism However, experiments which presumably result in adecrease in chalone concentration do not result in an increasein mitotic activity It is suggested that if chalones are physiological controllers of cell division they do not act by a simplenegative feedback mechanism but require the action of a substanceto decrease their concentration     

THE ROLE OF STRESS HORMONES IN THE CONTROL OF GRANULOCYTE PRODUCTION

It has previously been indicated that the inhibitory power of the granulocytic chalone is not influenced by adrenalin. It is now shown that this is true both in absence and in presence of exogenous hydrocortisone. It is also shown that hydrocortisone itself does not cause significant inhibition of DNA synthesis in rat bone marrow cells in vitro, but that it does act to augment the inhibitory effect which the granulocytic chalone induces. It is suggested that the primary action of hydrocortisone may be on the cell membrane which changes the cell wall permeability to chalone, perhaps by reducing its rate of loss from the cells.     

Effects of propranolol on mitosis-inhibiting activity of G2-chalone and adrenaline in cell culture of Ehrlich's ascites tumor]

Chalone-containing preparation from ascite Ehrlich's tumour blocks these cell transition from G2-phase to mitosis and its motion in mitosis in vitro (G2-block, M-block). Adrenaline blocks these cell transition from G2-phase to mitosis. Propranolol raises G2-block of chalone or adrenaline. Consequently this way of chalones action on cell division includes beta-adrenergic receptors influence of preparation on cell motion in mitosis doesn't change with addition of propranolol. Consequently this way of chalone system action on mitosis doesn't include beta-adrenergic receptors.     

Action of epidermal chalone on the vaginal epithelial cell proliferation in ovariectomized mice stimulated by 17 beta-estradiol

It has been shown that the DNA synthesis inhibitory effect of chalone on the vaginal epithelium of ovariectomized mice administered epidermal chalone three times (8, 4 and 1 h before 17-beta-estradiol injection) is dependent on chalone injection made 1 h before hormone injection. The decrease in the number of DNA synthesizing cells induced by 3-fold injection of chalone during 2 days is linked with the reduction in the level of exogenous estrogen in ovariectomized mice rather than with the duration of epidermal chalone action.     

Tissue-specific inhibition of cellular proliferation in Ehrlich ascites tumor]

The extract of cells of ascitic Ehrlich's tumour (chaloun) and its cell-free fluid produced a marked inhibitory action on the cell proliferation of this tumour four hours after the administration. The effect is tissue-specific, more pronounced in the extract and depends on the dose of the antigen. Eight hours after the extract or the cell-free fluid administration the mitotic activity in the tumour proved to increase in comparison with control; this indicated the presence of a short-lived chaloun action on the G2-phase of the mitotic cycle and synchronization of cell division.     

Effects of a lymphocytic chalone-containing preparation on the mitotic activity of mouse thymocytes in vivo

It has been shown that chalone-containing substance obtained from the rat spleen decreased the mitotic activity of mouse thymocytes upon its intraperitoneal administration to animals. The maximum inhibition was observed 45 min after the injection. Thus, the substance examined has the property of G2 chalone.     

Ehrlich ascites cell (EAC) chalone assay using purified calf thymus-DNA polymerase alpha

A relatively rapid chalone assay using inhibition of purified calf thymus DNA polymerase alpha by Ehrlich Ascites Cell (EAC) chalone has been performed. The DNA polymerase alpha was inhibited in a concentration-related fashion by partially purified EAC chalone ranging from 10 to 200 micrograms/ml. Spermidine was also tested since there has been some suggestion that chalone may be spermidine; we found no effect of spermidine at 170 and 230 microM, but marked inhibition at 33 mM. This assay should facilitate chalone purification, since chalone appears to non-specifically inhibit DNA polymerase alpha.     

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.     

A system for the study of epidermal G1-chalone in cell culture. The rat tongue epithelial cell line RTE2

A pig-skin preparation enriched in epidermal G1-chalone when administered to cells of the rat tongue epithelial line RTE2 at concentrations of 3-300 micrograms/ml (dry mass) caused a 60% reduction in cell number. Three other cell lines showed essentially no growth inhibition during chalone treatment. The kinetics of chalone inhibition were similar to those observed in mouse epidermis in vivo. Five hours after the addition of chalone preparation in fresh medium a decrease in the rate of DNA synthesis was observed. Maximum inhibition at 12 h was followed by a subsequent increase in DNA synthesis, reaching control values again after 30 h. The inhibitory effect was dose-dependent up to 3 micrograms/ml. At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 micrograms/ml. Removal of added chalone by changing the medium at the time of maximum inhibition gave rise to a complete recovery within 9 h. These results indicate a cell-line specific, non-toxic and reversible inhibitory effect of the chalone preparation which resembles that observed in the living animal. The RTE2 cell line may thus be considered to provide a highly sensitive experimental system suitable for more detailed studies on the mechanism of action of epidermal G1-chalone.     

ERYTHROCYTIC CHALONE, A TISSUE-SPECIFIC INHIBITOR OF CELL PROLIFERATION IN THE ERYTHRON

Control of the rate of cellular proliferation in the erythron seems to be mediated by a tissue-specific mitotic inhibitor, termed the erythrocytic chalone. the function of this substance seems to be to prevent excessive proliferation of the erythrocyte precursor cells by means of a negative feedback and in terms of peripheral cell numbers.
The erythrocytic chalone is present in mature erythrocytes, from which it can be extracted by incubation in a chemically defined medium. It is also present in fresh normal serum and it is possible that in physiological conditions the factor is continuously liberated from mature erythrocytes into the surrounding plasma.
In the rat, in an artificially induced polycythaemia the concentration of the chalone in the serum is increased and this increment appears to be the sole cause of the enhanced inhibitory action of polycythaemic serum on the proliferation of the bone marrow cells in vitro.
The mode of action of the erythrocytic chalone seems to be to prevent the erythrocyte precursor cells from entering the generative cell cycle; the chalone thus regulates the production of erythrocytes by changing the 'proliferation efficiency' in the erythron.
So far, nothing is known about the chemical nature of the erythrocytic chalone. However, in gel filtration it is eluted in the same zone as the granulocytic chalone, its molecular weight thus being about 2000-4000.     

Certain Mechanisms regulating cell renewal in the gastric mucosa in peptic ulcer

A study was made of the effect of an aqueous extract of human gastric mucosa on the mitotic activity of the surface epithelium of the mouse stomach. The extract was found to exert a statistically significant inhibitory action. An extract from the mucosa of the stomach resected for duodenal ulcer exerted a more pronounced inhibitory action as compared with an extract from the stomach resected for gastric ulcer. This fact may be accounted for by a greater content of mature differentiated cells in duodenal ulcer. Tissue-specific action of gastric chalone is indicated by the absence of mitotic inhibition in the small intestinal mucosa.     

Recovery kinetics of epidermal cell proliferation after two intraperitoneal injections of epidermal extracts (chalone)

The cell kinetic effect of two intraperitoneal (IP) injections of 5 mg of crude lyophilized skin extracts given 12 h apart was assessed during the recovery period (5 to 52 h after last injection) by measuring epidermal labelling indices and the specific activity after tritiated thymidine (3HTdR) injection, and by determining the cell cycle phase distribution by flow cytometry. The skin extracts produced an epidermal chalone effect and inhibited both DNA synthesis and mitosis. A slow recovery took place from 5 to between 22 and 36 h after the last chalone injection. During this period the cell flux into DNA-synthesis and mitosis slowly recovered, but the exits were blocked and cells accumulated in the respective phases. The fluxes out opened up at the S phase about 22 h, and at the M phase about 30 h after the second chalone injection. A secondary inhibitory effect was observed at about 40 h, followed by a subsequent recovery to normal at 52 h. The similarity between the recovery kinetics after chalone and that observed after hydroxyurea (HU) is emphasised.     

THE ROLE OF GLUCOCORTICOID HORMONES IN THE CONTROL OF EPIDERMAL MITOSIS

It has previously been established that the epidermal chalone inhibits epidermal mitotic activity more powerfully in the presence of adrenalin, although adrenalin itself is not a mitotic inhibitor. It is now shown that in low concentrations hydrocortisone has little if any antimitotic activity, that when it is present together with chalone and adrenalin it does not markedly increase their antimitotic activity, but that it does act to prolong the mitotic depression which they induce. It is known that, without hydrocortisone, adrenalin rapidly escapes from epidermal cells so that the chalone action is weakened. It appears that the role of hydrocortisone may be to reduce the rate of adrenalin loss and thus to prolong the chalone-adrenalin activity. It is also shown that the rate of loss of adrenalin from epidermal cells is inhibited, though to a much lesser extent, in the presence of excess chalone.     

Adrenaline Increases Cyclic 3′5′-AMP Formation in Hamster Epidermis

CATECHOLAMINES probably influence cell proliferation by delaying cells in the premitotic phase^1,2. Bullough and Laurence found that crude skin extracts contained a tissue-specific protein (chalone) which inhibited epidermal cell proliferation and that the action of this extract was augmented by adrenaline³. They later found that adrenaline alone (0.00025 µg/ml.) reduced epidermal mitotic activity in mouse ears by about 50% in vitro⁴.