Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 X 1 0 6 cellslml through daily centrifugation and resuspension i n fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are i n a Go or early GI phase, resuming growth readily i n response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring i n the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated i n the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.
In previous work lthe relationships between liver regeneration in rats, age and carcinogenesis with azodyes were investigated ( 1,2). This is a report on further investigations with the emphasis on the medhanism contrnlling liver regeneration.In vitro experiments. In order to investigate the possibility of the existence of humoral factors involved in liver regeneration, blood sera from partially hepatectomized andcontrol rats were compared with respect to their action: a ) on the initial growth response of primary rat liver explants cultivated in vitro, and b) on the growth period of in vitro cultures of a strain of normal rat fibroblasts. In the first series 24 male rats, 6 to 10 months of age grouped in pairs, were used. The experimental procedure emplbyed on each pair was as follows: -4 partial hepatectomy consisting of the removal of the two main lobes of the liver was performed on one of the partners and a sham operation consisting of a laparotomy and manipulation of the liver on the other. Since hepatectomy reduces food uptake, the diet of the control was reduced in order that his food uptake would not exceed that of the hepatectomized rat. Twenty-four hours after the operation 6he animals were bled and the sera o'btained were tested within 48 hours. Liver from a male rat 3 weeks of age was used as test tissue. A small piece of the tissue was cut up in the fluids to be used as culture medium and explanted directly on the glass wall of roller tubes without the use of plasma or embryo extract. Six tubes containing 6 fragments each were set up for each
Many physical and chemical factors, without being carcinogenic themselves, are able to cause an augmentation of tumor production when properly combined with the action of a specific carcinogen. Physical factors shown to be capable of such an action are: mechanical irritation (1) and scalding (2). Chemical factors are: a basic fraction of creosote oil (3), turpentine and chloroform (4), croton oil and its active component, croton resin (5).The most important fact, with respect to the mechanism of action of these irritants, is that none of them is effective when applied before rather than after the specific carcinogen (6). They act only when the carcinogenic process has been started by a specific carcinogen and carried on up to a certain point. This point may be such that the histological picture of the tissue under consideration cannot be distinguished from the normal one (4).Valuable information concerning the effect of irritants may be gained by consideration of a series of investigations which deal with the tumor-promoting action of surgically induced cell regeneration. Wounds were produced by excision or cauterization of skin in areas previously treated with suboptimal doses of a carcinogen. The subsequent healing process was found greatly to augment tumor formation (4, 7). It seems probable, therefore, that the action of irritants is in some way similar to the action of traumatic factors in the sense that both induce cell regeneration.These findings were considered to indicate the existence of a stage characterized by the formation of ceils capable of forming tumors but not asserting
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