The induction of tumors in Rana temporaria with nitrosamines.

Dimethylnitrosamine (DMNA) and diethylnitrosamine (DENA) dissolved in tank water in a dose of 5 and 50 ppm, resp. induced tumors of liver (hepatocellular cancers and adenomas), and of hematopoietic system (hemocytoblastosis). DMNA induced neoplasms in 19 of 43 frogs (44.2%); the average latent period being 18.2 weeks, while DENA induced tumors in 41 of 94 animals (43.6%) within a mean latent period 19.7 weeks. Results of this investigation show the possibility to use anuran amphibians in shorttermed experiments in the study of nitrosocompounds carcinogenicity as well as a biological indicator for environmental pollution.

About the influence of halogenated pyrimidines on the in vivo-oncogenesis.

Reported are in vivo trials on Friend virus-leukemia (FVL), on methylcholanthren-induced fibrosarcomas of the mouse, and on amelanotic melanoma No. 3 of the Syrian hamster after treatment with halogenated pyrimidines, 5'-bromodeoxyuridine (BUDR) and 5'-iododeoxyuridine (IUDR) as to provoke genuine oncorna viruses. Till now, no prove exists that in tissue culture propagated C-type particles are really oncogenic. From the results demonstrated here the conclusion can be drawn that the chosen in vivo-conditions neither oncogens, possibly located in the cells, will be finished to complete oncogenic viruses, nor a spread and multiplication of dormant viruses will take place due to a provocation treatment of the hosts with halogenated pyrimidines. It seems, therefore, that the requirements to activate oncogens in vitro are absolutely different to those needed under in vivo conditions.

Morphological and histochemical properties of human embryonic cells transformed by Rous and polyoma viruses.

It is shown that human embryonic cells transformed by Rous sarcoma virus (stable cell line 23) and those transformed by polyoma virus (stable cell line P-2) are morphologically distinguished from the normal human embryonic cells. The mitotic activity of P-2 cells was 51% and the mitotic activity of 23 cells was 48%. While the mitosis activity of human embryo fibroblast was 28%. The duration of the mitosis of P-2 cells was 20 hours and that of 23 cells was 18 hr. The duration of the mitotic cycle of human embryo fibroblast was 18 hr. The G1 periods lasted 6 hours for both the cell lines; the S period of P-2 cells lasted 8 hr and the S period of 23 cells was 6 hr. Both the cell lines had a high content of RNA, DNA, protein bound SH-groups, and a high activity of acid phosphatase, acid RNAase and glucose-6-phosphatase. The content of glycogen, and acidic mucopolysaccharides, the activity of NADPH-tetrazolium reductase, succinic dehydrogenase of both the lines were the same as in normal human cells.

Notes on the mechanism of postvaccination immunity in Marek's disease.

The investigated 16th and 45th in vitro passages of non-pathogenic variant 83 of the Kekava strain Marek's disease virus have led in chickens to resistance to Marek's disease by introduction of the above-mentioned virus 14 days before application of pathogenic variant 55 of the Kekava strain Marek's disease virus. Simultaneous administration of both variants of the Kekava strains Marek's disease virus did not protect chickens from the disease. Presence in those variants of the Kekava strain Marek's disease virus of genetic markers manifesting themselves on passaging the virus in chicken fibroblast cultures created the possibility to investigate interrelations between them in the organism of chickens, utilizing in isolation of the virus the method of infecting cultures with chicken fibroblasts. The results of isolation of the virus from the blood cells of vaccinated chickens have shown that in their organism there is interference between those virus variants since the frequency of isolation of the pathogenic virus variant was 3-times lower than that of the apathogenic Kekava strain Marek's disease virus, and both virus variants persisted in various cells. After simultaneous administration of both virus variants to chickens equal amounts of the pathogenic and of the apathogenic Kekava strain Marek's disease virus were isolated from their blood cells. In that case also persistance of both virus variants in one cell may occur.

Benzo(a)pyrene free-radicals formation in the presence of butylated hydroxyanisole and their possible importance in carcinogenesis.

By the method of UV irradiation of benzopyrene in the presence of butylated hydroxyanisole, the benzopyrene EPR spectra was investigated and the possible mechanism by which the carcinogenic activity of benzopyrene in the presence of butylated hydroxyanisole is lowered, was studied.

Enzymes relevant to cancer chemotherapy II. Heterogeneity of rat kidney uridine kinase following chromatography on Sepharose 6B coupled to N4-(5-amino-Pentyl)-cytidine.

Using Sepharose 6B coupled to N4-(5-aminopentyl)-cytidine at least three different peaks of rat kidney uridine kinase activity have been found. Prior to affinity chromatography the enzyme was partially purified by ammonium sulfate precipitation. The elution was carried out with 0.15 M NaCl or with 0.012 M ATP. The importance of different forms of uridine kinase in eukaryotic cells for cancer chemotherapy with uridine and cytidine analogues is discussed.

Mitochondrial ATPase of Zajdela hepatoma. II. Mitochondria of Zajdela hepatoma contain less adenosine triphosphatase than mitochondria of rat liver.

The specific activity and the content of ATPase in mitochondria of rat liver and Zajdela hepatoma were compared. The specific activity of ATPase in sonicated mitochondria and in mitochondrial membrane fraction of rat liver was almost two times higher than the specific activity in the corresponding fraction of Zajdela hepatoma. Accordingly, the autovertin binding capacity of rat liver mitochondrial membrane fraction as well as the yield of F1-ATPase from this fraction were about two times higher than those of the mitochondrial membrane fraction of Zajdela hepatoma. The results show that mitochondria of Zajdela Hapatoma possess about half amount of ATPase present in rat liver mitochondria.

Cyclophosphamide induced sensitivity against avian RNA myeloblastosis virus in age-resistant hosts.

The effect of cyclophosphamide administration on survival of 4- to 7-week-old chickens as well as on induction of leukemia after avian myeloblastosis virus (AMV) injection was studied. The drug treatment alone did not cause any neoplastic effect in the birds during 4 months of observation. Immediate application of AMV to cyclophosphamide-pretreated age-resistant chickens induced acute myeloblastic leukemia in about 80 per cent of test animals. The sensitivity of chickens against AMV, induced by cyclophosphamide, had transient character only. When AMV was injected delayed, 3 or 10 days later, after the administration of the drug was completed, a rapid and pronounced increase of resistance was observed again.

Biochemical properties of endogenous rat C-type viruses.

The cellular restriction to persistence of inducible C-type viruses of at least two rat strains can be overcome in actively in vitro replicating and/or spontaneously transforming rat embryo cells. Both, SD-RaLV and W-RaLV particles are morphologically and biochemically related to known oncorna viruses: They are of C-type, have an identical buoyant density of 1.15 g/cm3 in sucrose gradients and contain high-molecular-weight RNA. The RaLV-associated DNA polymerase exhibit both exogenous (polynucleotide-templated) and endogenous (viral RNA-templated) reactivity. The enzyme strongly preferred Mn++ over Mg++ ions (Mg++/Mn++ ratio of 0.04 to 0.07), with maximum reactivity at 0.1 mM MnCl2 concentration. The molecular hybridization experiments revealed the endogenous nature of the SD-RaLV and the W-RaLV. From the competition hybridization assay the number of proviral sequences could be calculated to be 84 per haploid cell genome. The role of endogenous rat viruses in the "spontaneous" in vitro transformation is discussed.

Significance of preoperational irradiation in the treatment of Wilms' tumor of the kidney (nephroblastoma).

Mitochondrial adenosine triphosphatase of Zajdela hepatoma. III. Effect of uncouplers on the hydrolysis of intramitochondrial ATP.

Hydrolysis of extramitochondrial ATP by coupled Zajdela hepatoma mitochondria is not stimulated by uncouplers of oxidative phosphorylation. The results of the present study show that the hydrolysis of intramitochondrial ATP in these mitochondria is stimulated by DNP and CCCP. It is proposed that the uncoupler insensitivity of ATPase in coupled Zajdela hepatoma mitochondria with exogenous ATP as a substrate result from an altered functional relationship between ATPase and ADP, ATP translocase.

Planimetric evaluation and comparison of roentgenograms of osteogenic sarcomas induced by 226Ra and 224Ra in mice.

Osteosarcomas were induced in mice by three activities of 226Ra (8.8 micronCi, 24.6 micronC and 70.5 micron Ci/kg) and by three activity levels of 224Ra administered in fractionated doses during a period of 75 weeks (total activity per mouse: 0.65 micron Ci, 1.88 micron Ci). In the 226Ra series 74 cases and in the 224Ra series 89 cases of osteosarcomas were evaluated by means of planimetric measurements of roentgenographic images, including the calcified structures. Even though it is not possible to differentiate the series precisely, still some differences between tumors under study could be found. In comparison with 226Ra series, the sarcomas induced by 224Ra are in average rather larger, calcified stuctures appear not to be fully developed and differentiated, he anatomical distribution is different from that characteristic to the 226Ra induced tumors, the mean life span is shorter. It is quite likely that not only the incidence but also some of the features of osteosarcomas are dependent on the time and tissue distribution of the dose delivered.

Presence of complement-fixing antibodies against antigens of Gross virus-induced rat lymphoma and normal rat thymus in sera of patients with some forms of malignancies.

Presence of complement-fixing antibodies against soluble antigen(s) of Gross virus-induced rat lymphoma W/Fu(C58NT)D was determined in sera of 180 healthy donors, in sera from 100 healthy pregnant women and in sera obtained from 139 patients with acute myelosis, acute lymphadenosis, acute undifferentiated leukosis, chronic myelosis, chronic lymphadenosis and Hodgkin's disease. Antibodies against soluble complement-fixing antigen from W/Fu(C58NT)D rat lymphoma were found in 33 (5).5%) out of 64 sera of patients with acute leukemia, in 15 out of 60 sera of patients with Hodgkin's disease, in 17 (9.4%) out of 180 healthy donors and in 22 (22.0%) out of 100 sera from healthy pregnant women. None of 15 sera from patients with chronic leukemia were positive. Titers of complement-fixing antibodies in positive sera remained unchanged after absorption with pooled lymphocytes from healthy human donors. Some selected positive sera reacted also with a soluble antigen prepared from normal rat thymus. Nature of complement-fixing antibodies detected and of corresponding antigens is discussed.

Experimental chemotherapy of rat leukemia RBA-Le with cis-diamminedichloroplatinum.

The antileukemic activity of cis-diamminedichloroplatinum (PDD) was studied in rats bearing myelogenous leukemia RBA-Le. Clinical picture, changes in life-span, hematological indices and weight changes were used to assess the effectiveness of the therapy. Maximum effectiveness was noted when PDD was given in combination with Methotrexate (MTX) and Poly I:C, respectively. The mean life-span of the rats treated with PDD and MTX was prolonged to 37 days that is an increase of 147 percent of the control level. Furthermore 20 percent of the treated animals survived symptom-free for more than 60 days. Out of 20 animals receiving PDD in combination with Poly I:C 6 rats survived 60 days symptom-free. In remaining 14 animals who finally died on leukemia an 85 percent increase in life-span was noted. Only slight increase in life-span was recorded in those groups treated with PDD alone and in combination with Cyclophosphamide regardless of dosage schedule. The general toxicity of PDD therapy is discussed.

Immunological response of patients with tumors of the urogenital tract: a follow-up of cell-mediated antitumor immunity by the LAI test.

Antigen-induced inhibition of leukocyte adherence is a suitable method providing information on immunological response of the tumor bearers. Leukocytes from patients' blood were tested by an in vitro method to detect cell-mediated antitumor immunity. Positive reactions of leukocytes to the specific antigen were found in carcinoma of the uterine cervix, vulva and ovary in 13 patients. The modified LAI test evaluated on the basis of free cells in Eagle's medium using the specific antigen is a simplified reversed method which, however, keeps the specificity.

Analysis of the results of treament of lung cancer patients with BCG according to the scheme of application of the bacillus.

BCG has successfuly been applied in lymphoid and in myeloid leukemia but no positive results have been demonstrated in solid tumors except malignant melanoma after intratumoral injection. The authors' approach consists in applying BCG as an only antitumor treatment, much smaller doses and in much longer intervals between them than used by other investigators. The analysis of the treatment schemes of BCG, applied to 171 lung cancer patients showed that positive responses have been obtained only in patients without peripheral dissemination of the disease and that in doses from 0.0001 mg to 0.05 mg. The results obtained depended on the frequency of BCG application: the 5 years survival rate, the mean survival period and the rate of the marked X-ray regression have been found best in patients treated once, good in patients treated in intervals longer than 30 days between the applications of the mycobacteria and worse in patients treated in intervals shorter than 20 days. A direct inverse correlation has been discovered between the mean survival period of the treated patients and the number of the applications of BCG in the first 6 months of the treatment.

Clinical experiences with Penberol (cis-beta-4-pentoxybenzoyl-beta-bromoacrylic acid) administered to patients with advanced neoplasias.

Kinetics of thymus- and bone marrow-derived lymphocytes in rats during tumor growth.

The kinetics of T and B lymphocytes were studied in the spleen, the lymph nodes, peripheral blood and the thymus of tumor-bearing rats in a time dependence following tumor transplantation with varying doses of allogeneic tumor cells. The dose of the transplanted tumor cells proved to be a limiting factor in the altered proportions of T and B cells in the various phases of immunological response of the tumorous host. A lower dose of tumor cells induced but a nonsignificant decline in the numbers of T lymphocytes in correlation with tumor growth and the tumor rejected within 19 days. A high dose of tumor cells caused an evident exhaustion of T cells in all the lymphoid organs followed (excepting the thymus), and the tumor was not rejected even by the end of the experiment. Evidently, the presence of tumor cells did not affect the B cell pool. The results are interpreted in the sense of a functional heterogeneity of the various subpopulations of T lymphocytes. The reactivity and responsibility of these subpopulations in the phase of tumor growth and its rejection is discussed.

DMBA-induced fibrosarcoma in bone. Histochemical and electron microscopical studies on new transplantable bone tumor of 7,12-dimethylbenz(alpha)anthracene-induced fibrosarcoma in rats.

Solubilizing effect of Trition X-100 on melanosome tyrosinase in hamster pigmented melanoma.

A study was made on the effect of Triton X-100 in different concentrations on the tyrosinase activity of two differently pigmented melanosome fractions of pigmented hamster melanoma, obtained by gradient centrifugation. Release of considerable tyrosinase activity was established spectrophotometrically in the supernatant of the treated melanosomes. The release of enzyme activity after treatment of the melanosome is probably due to a change in the permeability of the melanosome structures under the effect of the non-ionic detergent Trition X-100. A direct activating effect of Trition X-100 on the particulate enzyme should be excluded, since inhibition of DOPA-oxidase activity was established in the soluble tyrosinase fraction.

Human mammary gland associated antigens. Lack of D-type retravirus-induced antigens.

Antisera against tumor-associated and organ-specific antigens of human mammary glands, as well as mammary carcinoma patient sera were used in cross-reactions with the test-system for a nucleoid antigen of Hep-2 retravirus. None of the antigens, revealed in mammary carcinomas and human milk seems to be either viral structural, or virus-induced cellular antigen. No cross-reactivity was revealed between preparations, containing A-type particles, isolated from Hep-2 cells and human milk and mammary carcinomas. Cell surface antigen, nonidentical to viral structural antigens, was detected in Hep-2 cells. It proved to be shared by E16b and some other stable cell lines, however it cannot be detected in mammary fibroadenomas, positive for the D-type nucleoid antigen, and in mammary carcinomas. Only gastric carcinoma homogenates exhibited reaction of identity with the cell surface antigen, shared by Hep-2 and E16b cells. It suggests that some common stem cells might be involved in its synthesis, rather than D-type retravirus genome. This question is worth a special investigation. The findings obtained show the genome of D-type retravirus is repressed in mammary carcinomas in contrast with fibroadenomas, when synthesis of nucleoid antigens was revealed by ILYIN [7].

Complex of IgA1-lambda paraprotein and albumin.

The complex of IgA1-lambda paraprotein and albumin was analysed and its pattern was demonstrated in electrophoresis, immunoelectrophoresis, gel filtration and sedimentation analysis.

Pathology of tumors developed in guinea pigs given intraperitoneal injections of N-methyl-N-nitrosourea.

The carcinogenic effects of N-methyl-N-nitrosourea after repeated intraperitoneal administration in inbred strain NIH 13 guinea pigs were studied. 50% of the animals that survived beyond 22 weeks, after intraperitoneal injection of MNU at a dose of 10 mg/kg body weight/week for 18 weeks, developed a broad spectrum of tumors: these included adenocarcinoma of pancreas in 2 animals, fibrosarcoma of the mesentery in 2 animals, angiosarcoma of mesentery in 2 animals, mesothelioma of the peritoneum in 1 animal and tumors of small intestine in 3 animals. These studies indicate the susceptibility of different types of tissue to the local effects of MNU after intraperitoneal administration.

Results of dynamic dose-fractionation combined with breathing oxygen in comparison to conventional treatment in head and neck and uterine cervix cancer.

A dynamic dose-fractionation regime combined with breathing oxygen at ambient pressure was introduced in the treatment of head- and neck cancer, and uterine cervix cancer. The efficacy of the method was analyzed in relation to the results obtained by treating comparable cases with conventional fractionation regime. Final evaluation of the method will have to await the accumulation of further experience during a prolonged observation period. Encouraging results obtained so far suggest that the new treatment can be accepted as a possible improved routine in the therapy of some cancer sites.

The carcinogenicity of N-nitroso compounds formed endogenously in mice from benzimidazole carbamate pesticides.

Malignant lymphomas were developed in Swiss mice after intragastric treatment with carbamate pesticides combined with sodium nitrite in their drinking water. Attention is directed to the carcinogenic effect of N-nitroso compounds formed from carbamate pesticides in vivo. The importance of further investigations is stressed, since these results indicate that, under appropriate conditions, commercially available pesticides may increase tumor frequency in mice.

Effect of methotrexate sorbed on modified 2-hydroxyethylmethacrylate carriers in mice of C3H strain with a solid Gardner lymphosarcoma.

A aminohexylderivative of 2-hydroxyethylmethacrylpolymer has been prepared in the form of a microporous sponge, and Methotrexate (MTX) has been bound to this carrier by chemical sorption. Implantation of the carrier with sorbed MTX resulted in a decrease of tumor weight, while an implantation of the carrier alone or an i.p. injection of an identical dose of MTX had no effect on tumor growth. Tumor regression was found to be greater than after a repeated injection of MTX in a fourfold dose directly into the tumor.

Cancer mortality in a region with endemic nephropathy.

Cancer mortality in endemic and non-endemic villages from a region with endemic nephropathy (EN) of Vratza district, Bulgaria has been compared for the period 1965--1974. The total and for all cancer sites mortality rates in all studied villages were comparable to European and world standards except a very high mortality from tumors of urinary organs in the endemic villages. Age-adjusted to world population rates/10(5) population in hyperendemic villages have been estimated as 16.8 for males and 14.9 for females in case of kidney tumors and as 7.1 in males and 10.2 for females in case of urinary bladder tumors. On village group basis a very close correlation between mortality rates from urinary system tumors and EN incidence rates has been disclosed although endemic and non-endemic villages are neighbouring and are very similar in ecological and cultural terms.

Prognostic significance of skin reactivity in patients with bronchogenic carcinoma grouped according to the TNM classification.

Untreated patients with bronchogenic carcinoma of the epidermoid type showed a marked depression of cutaneous delayed hypersensitivity reactions to DNCB, PPD and Varidase. As compared to a control group of healthy individuals and to a control group of patients with non-malignant chest diseases, 46% of cases responded to the DNCB skin test, 56% to the PPD skin test and 39% to the Varidase skin test. The patients were subsequently divided according to the TNM classification in stage I, II and III groups. Correlation of the skin test positivity to the stage of the disease and to the survival of patients was followed.

The use of serial determination of blood serum glycoproteins for indirect evidence of the sensitivity and resistance of malignant tumor cells to clinical chemotherapy.

Sialic acid and mucoprotein tyrosine (MPT) as two important and sensitive indicators of blood serum glycoprotein changes were used for the dynamic follow up of the tumor growth in humans. Their determination was performed before and during chemotherapy with the aim to evaluate indirectly the sensitivity or resistance of malignant cell to the clinical chemotherapy. A total amount of 16 cases with gynecological malignancies was investigated. According to the results of the laborabory findings the patients were divided into three groups: a) with decreased values after chemotherapy, b) with no changes or increased values, c) uncertain results. The laboratory findings corresponded well with the clinical course of the disease.

Cytotoxic activity of macrocyclic metabolites from fungi.

The cytotoxic effect of 7 macrocyclic fungal metabolites on HeLa cells has been determined. The authors noted great differences in the cytotoxic activity of the substances investigated. Least effective had proved the metabolites curvularin and zearalenone which kept exhibiting ED50 at a concentration of 2 X 10(-4)M and at a concentration of 7.5 X 10(-5)M respectively, whereas the most effective cyanein and monorden gave rise to 50% inhibition of multiplication of HeLa cells at a thousandfold lower concentration than zearalenone. Cytochalasins A, B and D brought about 50% inhibition of proliferation of HeLa cells at concentrations of the same order, with ED50 ranging between 1.2-2.1 X 10(-6)M. The different biological activity of the studied macrocyclic fungal metabolites is discussed in connection with their chemical structure.