There are many investigators who have studied the effect of liver and other tissue homogenates or tissue extracts on the mitosis of various tissue cells and proliferation. Mc. Junkin and Breuhaus,131 Walter, Allman and Mahler,14) Bromgvist,4) and Teir and Ravanti3) reported that these substances had the promoting effect upon mitotic activity of rat liver cells, while Breus, Subbarow and Jackson,15) Stichand Florian,16) Marshak and Walker,17) Werner18) and Malmgren and Mills19) were in an opposite position by stating that they had an inhibiting effect upon it. Wilson and Leduce23) reported the rate of mitosis in the livers of young mice was at first inhibited, then stimulated by the intraperitoneal injection of pulped liver of the mouse and of the guinea pig. The motives of these effects are not yet distinct, but generally explained under the relationship of promoting. substances and inhibiting substances.
Chin27) has already been successful in extracting the substances inhibiting the growth of Ehrlich ascites cancer and N.-F sarcoma of mice from rat livers, and Chin and Kim= indicated that such substances were contained generally in the normal organs and tissues, and Chin and Suk24) also reported that these various tissue factors generally inhibited cellular mitosis and proliferation in hyperplastic tissue.
Then, the authors have made the following experiments to investigate further the effect of such growth-inhibiting factors on normal tissue and on tumor-free tissue(liver and skin) of tumor-bearing animals.
Materials and Methods
With 50 albino rats weighing about 300 gins. each, as one pool, the tissue factors were extracted from liver and skin by the same method as that done by Chin and Kim.28) Namely, these two. organs from rats -liver and skin-were macerated separately in a Waring blendor, and four volumes of distilled water was added to the emulsion-like substance. The emulsion was filtrated, and filtrates were centrifuged in a model PRI International centrifuge at 15,000/rpm for 45 minutes. One half to one volume of ethyl alcohol was added to the supernatant fluid. The sediment was placed under paper electrophoresis in sodium barbital and barbital buffer at pH 8.6. The slow-moving fraction was treated with trypsin in the incubator at 37´C and pH 8, and the material was dialyzed against distilled water in a cellophane tube at 4°C for 72 hours. The substances in the cellophane tube were removed and precipitated by an equal volume of aceton. The precipitate was completely dried, and the extracted dry tissue factors were ready for injection as a 5 or I per cent solution in physiologic saline solution. These two kinds of tissue factors were injected intraperitoneally to the rats and mice, then the mitoses of liver and epidemial cells were counted, and these were compared to that of the control, which received the same volume of physiological saline instead of tissue factors.
The Ehrlich ascites cancer transplanted mice(solid form) were used as the tumor-bearing animals.
The rats and mice were used as the experimental animals and these were bred with stock -diet. The rats were divided into two groups. One group received successive intraperitoneal injections of 5 mg. and 15 mg. of tissue factor everyday; the other group was injected once, intraperitoneally with 50 mg. of tissue factor. The Ehrlich ascites cancer transplanted mice received successive intraperitoneal injections of 1 mg. of tissue factor everyday. The groups which were successively injected were sacrificed on 3rd, 6th, 9th and 12th days after having received the .initial injection of tissue factor. In the 50 mg. single injection group, rats were sacrificed on 3 rd, 6 th, 9 th, 20th and 30th days respectively. Four hours before slaughter, the colchicine was injected intraperitoneally at the ratio of 0.1 mg. per 100 gm. body weight of the animals. The liver and skin slices were removed from a certain part under ether anesthesia, and then fixed in Carnoy´s fluid -for three hours. After dehydration and clearing, the tissues were embedded in paraffin, cut in -sections five micron thick, and stained with hematoxylin and eosin.
Mitoses of liver were counted in one hundred fields on each slide with the help of 43 x objective lens and 10 x oculars, and cell counts of epidermal cells were made for ten fields on each slide -with the help of 1.25 mm. oil immersion objective lens and 15 x oculars.
Results
1) Effect of tissue factors on the mitosis of normal rat livers showed that:- in the rat group which received the intraperitoneal injection of 5 mg. of liver tissue factor everyday, a marked inhibition of liver mitosis was observed in the early stages, and the inhibited mitosis--rapidly :reverted to normal; but it was also observed that it was apt to increase in the later stages (Tab. 1). In the experiment of consecutive injections of 15 mg. liver tissue factor everyday, the decrease rate was observed to be more significant, and the recovery was slower(Tab. 2). In the 50 mg. single injection group, action showed more outstanding inhibition of longer duration, at the same time, this inhibition seemed to recover to normal after many days(Tab. 3).
Effect of skin tissue factor on the mitosis was not significant,
2) Effect of tissue factors on the proliferation of epidermal cells of normal rat skin showed that in the experiment in which 5 mg. of skin tissue factor was injected consecutively to each rat everyday; there was no apparent effect on proliferation of epidermal cells of normal rat skin in the early stages, but in the later stages an increasing tendency in number of epidermal cells was observed(Tab. 4). In the experiment in which, an injection of 15 mg. skin tissue factor each rat received consecutively everyday, there was a further increase in number of epidermal cells in the` later stages(Tab. 5). On the other hand, in 50 mg. injection group of skin tissue factor, a decreasing tendency of it was observed in the early stages, whereas an outstanding increase was seen after many days in the later stages(Tab. 6).
In this study, there was no distinct effect of liver tissue factor, on proliferation of epidermal ,cells of normal rat skin.
To sum up, it is speculated that inhibiting factors have tissue specificity; when small amounts of tissue factor were injected successively, and the same organ tissue factor was applied to the same
organ, mitosis of liver and proliferation of epidermal cells were inhibited in the early stages, recovery was relatively rapid, and in the later stages there was observed an apparent promoting of mitosis and proliferation, even though the total dosage of tissue factors given was larger than 50 mg. dosage. Comparing the reaction in cases which received small amounts of tissue factor consecutively, and cases injected with a large amount !at one time there was a severe reaction observed in the latter. That is, in the early stages significant inhibiting action was seen, with a tendency to postpone recovery, but in the later stages, a marked promoting action was shown. It is also considered that these actions were seemingly strong in cases having been injected with liver tissue factor, and rather weak in cases which received skin tissue factor.
3) As a result of the comparative experiment(Tab. 7 and 8), mitotic cells of liver and epidermal cells in the tumor-bearing animals were counted, after they had been injected with 1 mg. of tissue factor everyday, successively, - and they were compared with that of the control which received saline injection only. The inhibiting effect of tissue factors on tumor-free tissue(liver and skin) of the tumor-bearing animals has generally been proved similar to that of normal animals, even stronger than normal animals, and slower to recover to normal range. This reaction was a little stronger in liver cells than in epidermal cells. It also revealed clear tissue specificity.
In conclusion, the inhibitory action of tissue factor to the normal liver and skin appeared to be weaker than to malignant and hyperplastic tissues, with remarkable promoting action in the later stages of the former. Consecutive injections of small doses showed more stable features than the single injection of the large dose, except in the initial inhibitory action. Reactions were tissue specific and stronger in the liver than in the skin. The inhibitory action of tissue factor to tumor-bearing animals was apparently more potential than that of normal animals, and this action was more apparent in the liver than in the skin.
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