We have recently found a familial series of colon cancers without polyposis. The characteristics of the family K are as follows. (1) Primary colon cancer affects three generations (five of 25 relatives had colon cancer). (2) The age of onset of cancer is about 20 to 30 years earlier than would be expected among the general population (ages ranging from 16 to 49 years). (3) All of the patients are male. (4) The proband was a 16-year-old boy, one of identical twins. The other twin shows no symptoms at present.
A total of 17 relatives in the family K was tested for lymphocyte blast transformation (PHA responsiveness). One of two affected members with colon cancer, one rectal polyp patient and 4 of 14 cancer-free relatives (29%) in the family K showed significantly decreased phytohemagglutinin (PHA) responsiveness in vitro (P<0.0025). When the age is limited to relatives over 20 years, which seems to be the high risk age for developing cancer in the family K, 5 of 7 relatives (71%) showed significantly decreased PHA responsiveness (P<0, 001).
This study clarified the HLA type in 21 relatives, including three relatives who died before this test, retrospectively. Nine relatives had A9-Bw35 haplotype and four of them (44%) had a current or previous colon cancer (relative risk=8.8). As for those over the age of 20, five persons had A9-Bw35 and four of these (80%) developed colon cancer. On the other hand, four relatives over 20 years of age did not have the A9-Bw35 haplotype and none of these had developed colon cancer. These observations suggested that immunogenetic factors might be involved in the development of colon cancer in members of the family K.

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The correlation of serum capacity for blocking and arming with effector cell reactivity has been examined in patients with breast cancer by using the leukocyte adherence inhibition (LAI) assay.
It was found that sera from breast cancer patients possessed the ability to make nonimmune mononuclear cells specifically reactive (i. e., armed) to the breast cancer extract and to specifically block the reactivity of mononuclear cells from breast cancer patients with the cancer extract. Sera from patients with breast cancer whose mononuclear cells were nonreactive to the corresponding tumor extract (6/8) showed higher blocking than sera from patients whose cells showed reactivity to the corresponding tumor extract (3/13). Similarly, sera from patients with positive LAI response (6/13) induced arming of normal mononuclear cells more often than sera from patients with negative LAI (2/8). There also appeared to be an inverse correlation between the capacities of serum from breast cancer patients to exhibit blocking and arming. That is, serum arming activity was found only in sera which showed no blocking, and serum blocking activity was detected only in sera without arming activity, except for one sample. The possibility is discussed that immune complexes play a key role in both serum activities, and that their biphasic character might be dependent on the ratio of antigen to antibody.

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A human pancreatic carcinoma cell line of islet cell origin (QGP-1) has been established and maintained for over two years. The parent tumor and the cultured cell line produce carcinoembryonic antigen (CEA), and there is no evidence of hormone secretion from the tumor cells. The epithelioid cells, which had migrated from rounded, irregular cell aggregates, grow as a confluent monolayer with piling up of cells in some areas, and have a population doubling time of 3.5 days. The modal chromosome number was 50. Exponentially growing cultures produce 76.3ng of CEA/10⁶ cells after 7 days. CEA production was confirmed by immuno-peroxidase staining.

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Various lots of cells obtained from Syrian hamster tissues were examined to determine their suitability for focus assay in chemical transformation in culture. Only a few lots of cells showed growth with sparse cell density during cultivation for 3 weeks without subcultivation, and not all lots of cells produced morphologically transformed foci after treatment with a carcinogen. Further, responsiveness to a tumor promoter, 12-O-tetradecanoyl phorbor-13-acetate (TPA), differed among different cell lots; cell lots which induced abundant growth consisting of small spindle-shaped multilayered cells upon treatment with TPA corresponded to those in which carcinogen treatment induced transformed colonies. When TPA was applied in transformation experiments, the morphologically transformed foci grew larger in cultures maintained in the TPA medium than in cultures kept in the TPA-free medium. We suggest that cell lots which show relatively sparse cell density at confluent culture and which show abundant growth consisting of small multilayered cells upon induction with TPA are suitable for transformation assay.

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The effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and other chemicals on the contact-feeding phenomenon were investigated. The death of 6-TG^r cells was prevented by TPA even upon culture with 3 times as many wild-type cells as in TPA-free medium. Seven out of 18 chemicals tested were shown to inhibit the phenomenon of metabolic cooperation. They were concanavalin A, lithocholic acid, chaetoglobosin A, 3-hydroxyanthranilic acid, urea, anthralin and saccharin. Among known tumor promoters, sodium deoxycholate, estradiol and phenobarbital did not inhibit the phenomenon of metabolic cooperation.

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Proliferation of normal human diploid cell strains was inhibited to a greater extent by treatment with theophylline than the proliferation of neoplastic human cell lines transformed in culture with Co-60 gamma rays (WI-38 CT-1) or SV40 (WI-38 VA-13). Theophylline was added to cultures at final concentrations of 1 to 3mM. Morphologically, normal fibroblasts became slender and small when grown in theophylline-containing medium, while little morphological change was observed in neoplastically transformed cells. Growth inhibition of theophylline was cytostatic rather than cytotoxic. No significant difference was detected in the uptake of theophylline between the normal and the transformed cells. Incorporation of ³H-thymidine into acid-insoluble fractions of cells began to decrease in the normal cells about 4hr after adding theophylline to the culture medium. The intracellular contents of adenosine 3', 5'-cyclic monophosphate increased linearly in the normal human cells with time of treatment with theophylline, but no increase was observed in the transformed cells. The present results suggest that the use of theophylline might be useful for quantitative experiments on neoplastic transformation in cultures of normal human cells.

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We have studied the chromosomal sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) of human diploid skin fibroblasts derived from individuals with adenomatosis coli (AC), which is a dominantly inherited disorder associated with multiple adenomas of the colon and rectum. Spontaneous frequencies of chromosome aberrations in the cell strains from the AC patients were similar to those in the control cells from normal individuals. However, the AC cells exhibited elevated chromosome instabilities when cells were exposed to MNNG, with aberration frequencies approximately twice as high as in similarly treated control cells. The present results, together with findings by others, suggest that the AC cells are defective in a function which regulates cellular condition and are in a state more susceptible to the action of agents that react with chromosomal DNA. These findings also raise the possibility of developing a diagnostic procedure for early detection of abnormal gene carriers of AC.

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Dichloro, dibromo, oxalato, malonato, dinitrato, sulfato and mono and bis(D-glucuronato) platinum (II) complexes of 1, 2-diaminocyclohexane (dach) isomers were prepared and tested on L1210 mouse leukemia employing the NCI protocol for evaluation of Pt analogs.
A large number of long-term survivors were observed with certain analogs, though the therapeutic indices (optimal dose/minimum effective dose) were not large. Among the analogs tested, the oxalato, malonato, dinitrato and mono-(D-glucuronato) Pt (II) complexes of trans-l-1, 2-diaminocyclohexane were found to be particularly effective. The glucuronato Pt complexes appear to be promising candidates for clinical trial since they have the highest solubility in water.

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One of the three kinds of mouse serum proteins increased in level by the administration of lentinan, designated LA, was found to be ceruloplasmin (p-diphenol: O₂: oxidoreductase; EC 1.14.18.1) which contains copper atoms and has amine oxidase activity. This mouse ceruloplasmin is a glycoprotein with a molecular weight of approximately 135, 000, and its chemical composition is very similar to that of human ceruloplasmin.

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Antiserum was raised in a rabbit against a particular AKR thymic lymphoma line, No. 8 (SAK 8), and was absorbed extensively with tissues of normal young AKR mice until it gave a negligible kill of normal thymocytes and lymph node cells in the presence of complement.
This absorbed antiserum (anti-SAK 8 reagent) plus complement was still cytotoxic for cells of the original SAK 8 tumor cells. The reagent also had relatively strong cytotoxic effects on RBL-5 and EL-4(G⁺) and weak effects on EL-4 (G^-) and E _??_ G-2, all C57BL/6 tumor lines. Absorption with E _??_ G-2 cells removed the cytotoxic effect of the reagent on E _??_ G-2 cells but not the effects on RBL-5 and SAK 8 cells. Absorption with RBL-5 or with EL-4 (G^{+, -}) cells removed all of the cytotoxic activity of anti-SAK 8 reagent, including that toward SAK 8 cells.
On the other hand, SAK 8 tumor cells were effectively killed by a goat anti-gp70 (Rauscher) antiserum and an anti-AKR endogenous virus antiserum raised in Fischer rats, but were not killed to a significant extent by a goat antip30 (Rauscher) antiserum.
The Fab fragment of anti-SAK 8 reagent inhibited the cytotoxic effect of anti-SAK 8 reagent on SAK 8 cells but did not inhibit the cytotoxic effect of the goat anti-gp70 or the rat anti-AKR endogenous virus antisera on SAK 8 target cells.
As in the case of other reports in which antisera raised against Moloney lymphoma detected Moloney cell surface antigen, which was different from virion antigens, the anti-SAK 8 reagent may be detecting a tumor-associated antigen(s) of AKR lymphoma cells that is different from the virion antigens and is crossreactive with cell surface antigens of C57BL/6 leukemias.

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In order to clarify the mechanism of therapeutic synergism elicited by a combination of 6-thioguanine and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), the cytocidal activity towards L1210 leukemic cells in vitro of these two drugs, alone and in combination, was examined by the soft agar cloning assay and the regrowth assay methods. More than 10-fold greater cell-killing activity than the expected additive survival (the product of fractional survival obtained for each drug alone) was observed on treatment with the combination of these drugs in both assay methods. Moreover, dose- and treatment schedule-dependent antitumor activity of this combination treatment against L1210 leukemia in vivo was clearly reflected in the cytocidal activity towards L1210 cells in vitro. These findings suggest that the therapeutic synergism elicited by the combination of ACNU and 6-thioguanine results from synergistic cytocidal activity towards tumor cells.

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In order to determine the mechanism of therapeutic synergism elicited by a combination of 6-thioguanine and 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) against various murine tumors, side effects on host animals due to the combination of these two drugs were examined. The combination toxicity index of these drugs given simultaneously was determined to be 1.5, i. e., this indicates that the combination of ACNU and 6-thioguanine is less-than-additive with respect to the lethal toxicity. Moreover, this combination treatment appeared to be at least no more than additive with respect to bone marrow toxicities as judged on the basis of bone marrow cellularity and peripheral white blood cell counts. In addition, the effects of treatment schedules, which were clearly observed in the therapeutic effect, on the side effects due to this combination treatment were also examined. However, no specific decrease or increase in side effects on host animals on this specific treatment schedule or the other treatment schedule was seen.

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The alkyl-nitrosourea-mediated formation of interstrand cross-links in E. coli DNA was studied by means of hydroxyapatite column chromatography. DNA interstrand cross-links were detected after treatment with 1-(2-chloroethyl)-3- cyclohexyl-1-nitrosourea (CCNU) but not after treatment with 1, 1'-ethylene-bis(1-nitrosourea) (EBNU) or 1-methyl-1-nitrosourea (MNU). Thus, alkylation of DNA by EBNU seems to be monofunctional, as with MNU.
The mutagenic and lethal activities of these three compounds were tested with a pair of Escherichia coli strains. EBNU gave slightly but significantly higher mutagenicity and lethality in a repairless (uvrA^-) strain than in its parental strain with wild-type DNA repair, whereas CCNU and MNU showed equal mutagenicity and lethality in the two strains.

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The expression of two markers of fetal liver, α-fetoprotein (AFP)and γ-glutamyltranspeptidase (GGT), and the relationship between these markers and tumorigenicity were studied in 17 continuous epithelial-like cell lines derived from normal rat livers and ascitic hepatoma cells.
Fourteen cell lines positive for GGT activity included 6 non-transplantable lines. Out of 10 cell lines transplantable to syngeneic rats and/or immunodepressed hamsters, 2 showed no activity of GGT cytochemically.
AFP production was seen in the culture fluid of 4 out of 15 cell lines tested. They consisted of 1 GGT-positive, transplantable, 1 GGT-negative, transplantable, and 2 GGT-positive, non-transplantable cell lines.
These results indicate that no correlation exists among these 3 parameters related to neoplastic and preneoplastic states of cells.

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A study was conducted to evaluate the antitumor activities of six nitrosourea derivatives against the xenograft of mammary breast carcinoma transplanted in nude mice (MX-1). The drugs employed in this study were 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), 1-(2-chloroethyl)-3-(methyl α-D-glucopyranos-6-yl)-1-nitrosourea (MCNU), 1-(2-chloroethyl)-3-(β-D-glucopyranosyl)-1-nitrosourea (GANU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (Me-CCNU), and, 2-[3-(2-chloroethyl)-3-nitrosoureido]-2-deoxyl-D-glucopyranose (DCNU, chlorozotocin). CCNU and Me-CCNU were administered intraperitoneally, and the others were given intravenously through a tail vein. Antitumor activities were assessed in terms of the drug-induced tumor growth inhibition based on caliper measurements. A single treatment with ACNU (40mg/kg) induced 92% tumor regression, compared to 73% and 69% tumor regression induced by MCNU (15mg/kg) and CCNU (50mg/kg), respectively. GANU and DCNU were less effective. To evaluate the antitumor activity of the drugs, we employed the predetermined dose lethal to one-tenth of BDF₁ mice (LD₁₀) for each drug as a standard therapeutic dose to nude mice; doses higher than LD₁₀ and one-half and/or one-fourth of LD₁₀ were also given. The results suggested that LD₁₀ in BDF₁ mice could be employed as a standard therapeutic dose in the chemotherapy of nude mice.

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The present study was carried out to investigate the preventive effect of Propionibacterium acnes (P. acnes) on metastasis formation in C3H/He mice which had been rendered tolerant to tumor-associated transplantation antigen (TATA) of syngeneic X5563 plasmacytoma. C3H/He mice inoculated intravenously (iv) with 10⁶ 7000 R X-irradiated X5563 tumor cells 3 times at 4-day intervals. These pretreated mice could not develop immune resistance against X5563 tumor even after the appropriate immunization procedure. In these TATA-tolerant mice, enhanced metastasis associated with the development of visually apparent metastatic nodules in the spleens was observed after intradermal (id) implantation with viable tumor cells, even if the tumor was resected surgically 7 days thereafter. Administration of P. acnes to these TATA-tolerant mice appreciably reduced the metastasis formation. Although the growth of the id primary tumor was not affected by injection with P. acnes, the combined treatments of surgical resection of the primary tumor and P. acnes administration resulted in complete protection against metastasis in TATA-tolerant mice. These results unequivocally demonstrated that the administration of P. acnes is effective for preventing the outbreak of metastasized tumors, which are readily inducible in the TATA-tolerant state.

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The effect of aclacinomycin A on the survival of cultured mouse L cells was studied. The drug showed a characteristic dose-survival response, i. e., an initial exponential response followed by a sigmoidal inactivation characterized by a broad shoulder. With prolongation of the exposure time, the exponential response seen at a low concentration was remarkably intensified with a progressive decrease of the shoulder width. Two-dose fractional survival studies revealed that at least a portion of the shoulder region related to an accumulation of sublethal damage which can be repaired during 12hr after the first dose. The implication of these results for therapeutic scheduling is discussed.

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In gastric cancer surgery, an extended radical operation is commonly performed, and in cases of total gastrectomy, there is a tendency to perform splenectomy at the same time. However, some surgeons have reservations regarding this treatment in connection with the possible preservation of the host resistance.
The question arises, is it reasonable to accept, by simple analogy with prophylactic lymphadenectomy the concept of the prophylactic splenectomy? The present study was designed to cast light on this problem.
In order to examine the value of prophylactic splenectomy in gastric cancer surgery, a comparable patient group was followed up, and it was found that the non-splenectomized group showed a significantly better late survival rate than the splenectomized group (P<0.05), the 4-year survival rate being 63% in the former group and 36% in the latter group. Although these results do not necessarily contraindicate combined splenectomy, it seems desirable to reappraise the value of prophylactic splenectomy in cases having no metastasis in the splenic hilar and adjacent lymph nodes.

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A cloning method utilizing the enzyme histochemical procedure was applied to isolate variant HeLa subclones which produce different alkaline phosphatase isozymes. Phosphate was used to distinguish between Kasahara isozyme and Regan isozyme. The use of filter paper made it possible to determine the localization of the Kasahara isozyme-positive colonies. By this method, two variant clones, HeLa S3-10KP and HeLa S3-10KN, were isolated. Kasahara isozyme was induced in the former by increased cell density, but not in the latter.

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In immunological studies, cytotoxicity assays are extremely important. The assay method was modified so that the radioactivity of beta-decaying isotopes in a large volume of aqueous sample could be counted. Through tests of various scintillators, it was found that emulsion scintillators had a higher counting efficiency than toluene- or dioxane-based scintillators. The results obtained by this method using an emulsion scintillator agreed well with those obtained by the ⁵¹Cr release method. The advantages of the emulsion scintillator method are as follows: (a) the activity of a beta-decaying isotope can be counted in a large volume of aqueous sample, (b) a long half-life beta-decaying isotope can be used, (c) the effects of free labeled materials can be eliminated by simple procedures, and (d) no gamma counter is needed.

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The titration curves of ecotropic murine leukemia viruses with various rat cells were determined by the reverse XC assay method. The curves of N-, B-, and NB-tropic viruses with Hepano cells (derived from a Sprague-Dawley rat) and Sprague-Dawley embryo cells were of single-hit type. In contrast, the curves of these viruses with 3Y1 cells (derived from a Fischer rat) and Fischer rat embryo cells were of two-hit type. Thus, the cells derived from the Sprague-Dawley rat were sensitive to infection by ecotropic murine leukemia viruses but the cells derived from the Fischer rat were resistant.

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The antitumor activity of 1-hexylcarbamoyl-5-fluorouracil (HCFU) in various schedules of long-term oral administration was examined in spontaneous mammary adenocarcinoma of SHN mice, an autochthonous tumor system. In the control group, the average time to local recurrence and average longevity after surgical intervention were 21 and 48 days, respectively. Oral administration of HCFU at 200∼300mg/kg/day, 3 times a week or for 5 consecutive days every 2 or 3 weeks was markedly effective against the adenocarcinoma. The optimal schedule was 20 administrations of HCFU at 300mg/kg/day, 3 times a week. The average time to local recurrence after the operation was increased to 200% and average postoperative survival was also prolonged to 150%. Growth of the tumors was slower and lung metastases at autopsy were found to be suppressed by HCFU. The effect of HCFU in delaying local recurrence and prolonging longevity was slightly affected by the schedule of administration.

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The effect of cyclophosphamide (CP) on the urinary bladder epithelium of rats treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was examined histologically to detect preneoplastic papillary or nodular hyperplasia. The urinary bladder epithelium of rats injected intraperitoneally with 40 or 80mg/kg body weight of CP after treatment with 0.025% BBN for 2 weeks showed extensive epithelial erosion and then simple, or papillary or nodular hyperplasia, but this soon almost disappeared. In contrast, rats given BBN alone showed papillary or nodular hyperplasia later. These findings indicate that epithelial damage by CP inhibited the early neoplastic process of BBN.

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