Micro-necrotic foci in regression of a murine calvarium transplantable tumor. 1. Cultivation and growth parameter of spontaneously transformed calvarium cell line.

The experiments reported in this communication describe the establishment of long-term cell lines, by in vitro cultivation of murine calvarium under: a) normal, physiological condition (Cont.Cal), where the cells were grown in standard Eagle's minimal essential medium (MEM), every 72 hours they were replenished with fresh medium and subcultured every 14 days, and b) experimental, non-physiological (Expt.Cal) condition, where the cells were grown in standard MEM, the cells were replenished with fresh medium every 28 days. During this period, and at weekly intervals the pH of the medium was readjusted to 7.3--7.5. These experimental cultures were subcultured once very 28 days at the time of feeding. Determinations of growth rate, requirement for serum growth factors, growth in soft agar, agglutination of the cells by lectin and their behavior on lectin columns, showed striking differences between the cells from the two conditions (Cont.Cal) and (Expt.Cal). It is suggested that either transformation or cellular selection occurred under the non-physiological conditions.

Micro-necrotic foci in regression of a murine calvarium transplantable tumor. 2. Enzymatic modification of the transformed calvarium cell immunological characteristics.

The experiments reported in this communication examined the immunological properties of cell lines cultured under (a) normal, physiological (Cont.Cal) and (b) experimental, non-physiological (Expt.Cal) conditions described in the preceeding paper. Determination of oncogenicity, humoral and cellular immunity of the cultures at various subcultures induced in syngeneic, unconditioned CFW1 mice, showed differences between cells from the two conditions. Cells grown under the experimental conditions (Expt.Cal) became oncogenic. Oncogenicity increased with number of subcultures, and treatment with vibrio cholera neuraminidase rendered the cells non-oncogenic. It is suggested that the experimental conditions either cause malignant cell transformation, or selection of oncogenic cell variants. Since oncogenicity is reduced by treatment with neuraminidase it could be related to certain cell membrane determinants.

Micro-necrotic foci in regression of a murine calvarium transplantable tumor. 3. Histological characteristics of the micro-necrotic foci.

The experiments reported in this communication correlate histiocytes and plasma cells infiltration into the implantation site of Expt.Cal cells, with formation of micro-necrotic foci and tumor regression. Subcutaneous implantation of any subculture from Cont.Cal cells produced lesions which disappeared 48 to 72 hours later. Cont.Cal cells from any subculture produced no tumors. After several subcultures and upon subcutaneous implantation of Expt.Cal cells lesions were formed which develop into solid tumors. The magnitude of oncogenicity of Expt.Cal cells increased with the number of subcultures. Histological examination of the site of implantation revealed actively proliferating fibrosarcoma cells. The characteristics of the cell population confirmed those of actively growing malignant cells. Multinucleated giant cells and Reed-Sternberg cells have been found in all the examined sections from implantation site of Expt.Cal cells. Treatment of oncogenic Expt.Cal cells with vibrio cholera neuraminidase (VCN) rendered the cells non-oncogenic. Inoculation of VCN-modified Expt.Cal cells induced an extensive histiocytes, lymphocytes and plasma cell infiltration leading to tumor cell destruction and formation of micronecrotic foci.

Cytogenetics of carcinoma of the endometrium. I. The study on ploidy.

98 patients suffering from carcinoma of the endometrium were cytogenetically examined with positive results in 31 cases. The majority of positive cytogenetic examinations of tumors showed hypodiploid stemlines with most frequently appearing modal number 44 chromosomes. Polyploid stemline was observed in 9 and in 2 cases. In the group of 20 well-differentiated carcinomas of the endometrium, 17 tumors hypodiploid and out of 9 poorly-differentiated carcinomas, 7 exhibited stemlines in the polyploid region. It is presumed that cytogenetic development of carcinoma of he endometrium leads from a diploid cell to a hypodiploid one and is terminated by the outgrowth of polyploid cell clones.

The particular traits of carcinogenesis induced in Wistar rats by aflatoxin B1. III. Porphyrins and the activity of gamma-glutamyltranspeptidase in primary hepatomas and in their tissue of origin.

Inbred Wistar rats were fed aflatoxin B1 (AFB1) during a period of 15 weeks. Some of the rats were sacrificed successively at different time intervals, and the remaining animals died a natural death. Beginning with the day of AFB1 treatment termination multifocal hepatomas appeared on the 265th day in males and on the 296th day in females. The progress of AFB1 carcinogenesis was accompanied by an ever increasing disorder of porphyrin metabolism. The content of the porphyrin fractions in primary hepatomas was greater than in the tissue of origin. Serum and hepatic GGTP activity increased with the advancement of hepatocarcinogenesis and paralleled the increase of tumor weight. The GGTP activity in hepatoma reached values exceeding those in their host liver.

Breast and other cancer deaths in a mental hospital.

The neuroleptics, well known as stimulants of prolactin release, are supposed to increase breast cancer incidence. To verify this hypothesis, we selected a group of 853 deaths of female inpatients with mental diseases recorded in the Dr. G. Marinescu Hospital between 1929 and 1978. Using proportionally and indirect standardization methods, we did not find any association between the incidence of breast cancer death and neuroleptic therapy, widely used in the above-mentioned hospital after 1959. Several authors reported a low cancer death incidence in mental patients. Between 1925 and 1978, 2168 deaths were recorded in this hospital. Besides death certificates, we also studied 1444 complete autopsy protocols (66.60% of all the deaths). Cancer deaths represented 1.94% of 1231 deaths recorded between 1925 and 1960. Cancer deaths represented 7.04% of 937 deaths in the period of 1961-1978, in comparison with 13.36% of the whole population of Romania (p less than 0.001). Among these 937 deaths, statistically significant lower cancer ratios than in general population were found in ten-year age groups, i.e. between 15 and 74 years in women and between 45 and 74 years in men. No case of leukemia was recorded over 1925-1978. Deaths from pneumonia, bronchopneumonia and cardio-vascular diseases are now frequent in major mental disorders. New prospective studies are required to elucidate the problem of cancer incidence in mental patients.

Karyotypes of the 5-azacytidine-sensitive and -resistant line of AKR mouse leukemia.

The karyotype of AKR mouse lymphoblastic leukemia cell line made resistant to 5-azacytidine was compared with the karyotype of parental line. In relation of 5-azacytidine-resistant line the parental cell have less chromosomes No. 5 and more of No. 17 per karyogram. The increased monosomy has been found in chromosomes No. 5, 6, 7, 8 and 18 in sensitive line and in chromosomes No. 1, 8 and 17 in 5-azacytidine-resistant cells. The highest occurrence of trisomy was present in the chromosomes No. 13 and 16 in both lines. In AKR sensitive cells 89% of karyograms displayed 3 to 13 markers per karyogram while in the resistant line there were only 37% of karyograms with 1 to 11 markers.

Studies on glucose-6-phosphatase, fructose-1,6-diphosphatase activity, glycogen distribution and endoplasmic reticulum changes during hexachlorocyclohexane induced hepatocarcinogenesis in pure inbred Swiss mice.

Inbred Swiss mice were fed hexachlorocyclohexane (BHC) at 500 ppm dose level in diet for 2, 4, 6 and 8 months. Later BHC was discontinued for 4 months and subsequently the animals were refed BHC for 1 month. Glucose-6-phosphatase (G6Pase) and fructose-1,6-diphosphatase (FDPase) activity was studied at different time intervals accompanied with changes in glycogen distribution and endoplasmic reticulum (ER) proliferation in hepatocytes. G6Pase and FDPase showed a decline in activities on BHC feeding. The activities of these enzymes showed recovery on BHC discontinuation. The changes were progressive with duration of exposure. After 6 months exposure the biochemical changes became more resistant to recovery. Maximal changes occurred in 8 month-exposure and the changes were irreversible. Glycogen accumulation and depletion followed a definite pattern. After two months of BHC feeding, increase in parenchymal glycogen storage zones was observed. In the later stage of hepatocarcinogenesis and specially in tumors, glycogen was depleted considerably. Smooth endoplasmic reticulum (SER) proliferation was recorded around the 3rd and 4th month. The correlation between glycogen accumulation, SER proliferation, G6Pase and FDPase activity is discussed.

Host range of murine sarcoma virus, its host modification and phenotypic mixing with endogenous virus.

The M(MSV)AG-50 cells produce competent ecotropic sarcoma virus which is able to transform several rodent embryo cells and replicate in them. The expanded host range has shown that the virus acquired some host information which facilitate the transformation of heterologous cells. The transformed cells were XC positive and with transformed phenotype in vitro. The genome of mouse sarcoma virus from nonproducer rat liver cells could be rescued by xenotropic endogenous virus. The obtained virus has shown the augmented host range for various embryo cells and for some mammalian cell lines as well. The virus with xenotropic coat efficiently transforms the cells, yielding cells with transformed phenotype. The majority of this virus were phenotypically mixed virions, with minority of probably recombinant virus as suggested by XC test. The modification of the virus during the passage through heterologous cells is discussed.

Demonstration of a syncarcinogenic effect of endogenous MuLV, rescued from MNU-induced leukemias, and suboptimal MNU doses in mice.

It was demonstrated that the combined action of activated endogenous MuLV isolated from MNU-induced leukemias and suboptimal doses of MNU yields a significantly high incidence of leukemias and tumors. Treatment with one agent alone shows low effects only. The findings were discussed in view of the mechanisms in which viruses and chemical carcinogens interact in a syncarcinogenic processes.

Establishment and characterization of permanent cell lines from patients with acute and hairy cell leukemia.

Four cell lines were established from peripheral blood of patients with leukemia. All lines express the Epstein--Barr virus nuclear antigen (EBNA) and therefore should be classified as lymphoblastoid cell lines. However, one of the lines UHKT-5 established from a patient with acute myelomonocytic leukemia has some features not typical for lymphoblastoid cell lines. The cells resemble to macrophages with the phagocytic ability for yeasts, strong alpha-naphthylacetate esterase positivity in phagocytizing cells and unusually long villi. Another line UHKT-7 established from a patient with hairy cell leukemia expresses the same isotype of membrane immunoglobulins as the original hairy cells.

In vitro studies of a co-carcinogenic effect of vaccinia and herpes group viruses.

The results of studies of a co-carcinogenic effect of two human infectious viruses in tissue culture are reported here. Viable vaccinia virus actively replicating in the cells of primary BALB/c tissue culture and in a number of continuous murine cell lines has been shown to induce in them expression of major structural p30 protein of murine retroviruses. Vaccinia virus has been also shown to cause biochemical transformation of murine cells. Evidence for the capacity of herpes simplex virus type 2 to induce malignant transformation of BALB/3T3 murine cell line has been obtained and confirmed by transplantation to mice. Transformed cell clones did not contain complete infectious herpes simplex virus but were resistant to superinfection with this virus. N-tropic endogenous murine retrovirus of C type with buoyant density in the saccharose density gradient of 1.18 g/cm3 and a reverse transcriptase activity was expressed in the transformed cells. In the virion structure six proteins typical of these viruses with the prevalence of p30 have been demonstrated. Competitive radioimmunoassay revealed a very high level of virus production: p30 level reached 7500 ng p30 R-MuLV per mg of viral protein. Specificity of this results was shown in control experiments.

The expression of virus-specific nucleotide sequences in Rous sarcoma cells.

Poly(A)-enriched virus-specific mRNAs (v-mRNA) are revealed in the subcellular fractions (nuclei, mitochondria, polyribosomes and cytosol) from chicken and hamster Rous sarcoma cells. The qualitative and quantitative differences in v-mRNA populations from permissive and non-permissive tumors are shown. The sedimentation analysis of v-mRNAs allowed to ascertain the presence of three molecular classes of v-mRNA in nuclei and cytoplasm of chicken Rous sarcoma cells (35S, 28S and 22S, and 35S, 26-28S and 20S, respectively). While two size classes (33S and 20S) are shown in hamster Rous sarcoma nuclei, only 24S v-mRNA is revealed in the cytoplasm of its tumor tissue. Thus, the oncovirus gene expression is restricted in the non-permissive tumor cells, unlike of the permissive chicken Rous sarcoma cells, both in the level of transcription, and in the processing and transfer v-mRNAs from nucleus to cytoplasm. The virus gene function peculiarities in the tumors of birds and mammals are discussed.

Calculation of reactivity indices for benz(a)anthracene and benzo(a)pyrene and their approximative models of complex with enzyme epoxidase.

By theoretical methods of quantum mechanics an interaction between carcinogenic hydrocarbons -- benz(a)anthracene, benzo(a)pyrene -- and various groups simulating an effect of enzyme oxidase has been studied. If accord is to be achieved with formation of epoxides of these compounds in vivo according to the bay region theory (i. e. in A region) an interaction between the enzyme involving an electron-acceptor group and the bay region of hydrocarbon under simultaneous steric hindrance of the K region is necessary. Another possibility of obtaining the highest reactivity in the A region is to assume the formation of the radical cation as a first intermediate in the interaction with enzyme.

Organization of proviral DNA and protein composition of hormonally stimulated C57Bl/10 strain-associated mouse mammary tumor virus.

Two continuous lines, BL-MaTU/A1 and BL-MaTU/s6, were established from C57Bl/10 mammary adenocarcinomas induced by DMBA-prolactin-estradiol treatment. Under in vivo stimulation with dexamethasone, insulin, prolactin, and prostaglandin A1, the cells produce detectable amounts of B-type particles with biochemical properties similar to the GR-MuMTV. Analysis of restriction endonuclease-generated fragments of cellular DNA revealed identical patterns in the integration sites and internal recognition sites of MuMTV proviral equivalents in the tumor cells and normal organs of C57Bl/10 strain mice. The restriction DNA fragments of C57Bl/10-associated MuMTV proviral DNA are closely related to those of the Balb/c-associated MuMTV. These results indicate the endogenous origin of MuMTV produced in the hormonally stimulated cultures of DMBA-prolactin-estradiol-induced C57Bl/10 mammary tumors.

Diagnosis of bovine leukosis virus infection by solid phase radioimmunoassay for virus antibody.

Solid phase radioimmunoassay for detection of anti-BLV antibodies in sera of infected animals is described. Viral antigens bound to surface of polystyrene are able selectively adsorb the antibodies from serum. Detection of bound antibodies quantitatively was done by 125I-labeled protein A from Staphylococcus aureus. Technical details of the assay are reported. The assay could be used as competitive one as well. Two different methods for detection of anti-BLV antibodies were compared with solid phase RIA. The solid phase RIA is highly sensitive, specific and available for large-scale examination. The assay is recommended for early diagnosis of bovine leukosis.

Occurrence of two distinct subpopulations of suppressor cells in rats bearing chemically induced tumors.

Spleen cells from rats bearing syngeneic methylcholanthrene-induced sarcomas showed impaired proliferative responses to phytohemagglutinin (PHA) as well as to the KCl tumor extract. Studies were then performed to define suppressor cell systems possibly operating in tumor-bearing rats. For this purpose, thymus and spleen cells from tumor-bearing rats were admixed with tumor-immune or normal rat spleen cells and were assayed for their suppressing activity on proliferative responses of immune spleen cells to the tumor antigens and on those of normal spleen cells to PHA. Anti-rat T cell serum and glass-adherent technique were utilized to characterize the cellular source of suppressor activity in the thymuses and spleens of tumor-bearing rats. The results indicate that there are two distinct subpopulations of suppressor cells in tumor-bearing rats. The first is glass-adherent non-T suppressor cells that inhibit lymphocyte proliferative responses both to the tumor antigens and to PHA. The second is suppressor T cells and can only impair proliferative responses of tumor-immune lymphocytes to the tumor antigens. The former cells were found in tumor-bearers' spleens, while the latter cells were present in their thymuses and spleens. It is suggested from these data that complex mechanisms of immunosuppression are working in tumor-bearing hosts, one of which can be attributed to the existing suppressor cells in those host lymphoid tissues.

Tumor promoting and skin irritant diterpene esters of Euphorbia virgata latex.

The mouse ear irritant latex of E. virgata, has been found to contain various skin irritant and non-irritant esters of the diterpene ingenol. In cocarcinogenic assay on dorsal back skin of NMRI mice, acetone extract of this latex produces squamous cell papilloma. Using a combination of biologic and chemical separation techniques one of the initiator-promotor for skin carcinogenesis has been identified as 3-0-2 methyl-decanoyl ingenol.

Toxicologic and therapeutic examinations with ftorafur in different mouse strains.

Ftorafur, a derivative of the antimetabolite 5-Fluorouracil, was tested for its toxicologic and therapeutic effectivity in the mouse strains XVII/Berlin, F1 (NMRI X DBA/2), Balb/c -- +/+, Balb/c -- nu/+, Balb/c -- nu/nu. The LD values were similar in all strains tested, with a somewhat higher sensibility of the homozygous nude mice. In each case lethality was higher in male than in female animals. This tendency was confirmed by the always more pronounced leukopenia in male versus female mice after high doses of Ftorafur. Thrombocytes, hemoglobin and hematocrit were not influenced by Ftorafur treatment, whereas marked reticulopenias were observed. While in the L1210 model significant ILS values without distinct leukopenias were obtained, no convincing effectivity could be demonstrated in any of the other tumor models. The results confirm that it is possible to compare phenotypical normal with nude mice both with regard to dosage levels and therapeutic activity.

Cytophotometric and cytogenetic characteristics of primary hepatomas induced in Wistar rats by aflatoxin B1.

Karyograms of the primary hepatomas induced in Wistar rats by aflatoxin b1 exhibited hypodiploid chromosome sets, and destruction of the chromosomes. Cytophotometric investigations showed a decrease of DNA content in the neoplastic cells, even to about 40% of DNA content in the normal hepatocytes in rats. In the neoplastic destruction of the chromatin material in these hepatomas breaks and deletion of the chromatids predominate.

Some antigenic similarity between nonhistone protein-DNA complexes from chromatin of the cells of Zajdela hepatoma and liver of rats subjected to a single 4-dimethylaminoazobenzene injection.

A comparative study of the antigenic properties of the nonhistone protein (NHP)-DNA complexes, isolated from liver and kidney chromatin of intact rats and from Zajdela hepatoma cells, revealed in the latter the presence of an antigenic component characteristic of kidney chromatin but never occurring in the liver chromatin of intact rats. A similar antigenic component is found in rat liver 4--7 days following a single injection of the hepatocarcinogen 4-dimethylaminoazobenzene and is not observed after an injection of the non-carcinogenic analogue 4-diethylaminoazobenzene. It is noteworthy, that this chromatin alteration resulting from carcinogen treatment is temporary, since on the 11th day following such treatment the kidney antigen is no longer found.

Dopa oxidase activity and ceruloplasmin in the sera of hamsters with melanoma.

Two simple spectrophotometric assays have been employed for the measurement of dopa oxidase activity and ceruloplasmin polyphenol oxidase activity in the sera from normal hamsters and hamsters bearing melanotic melanoma. Both activities were found to be augmented in tumor animals, the dopa oxidase activity much more prominently. The levels of the enzymes tested increased proportionally to the tumor mass.

Clastogenicity and sister chromatid exchange induction by ftorafur.

The main effect of Ftorafur at the chromosomal level is the induction of chromatid and chromosome breaks, which is some pronounced in neoplastic or transformed cells than in normal cells. Different cell lines used in the study exhibited both in vitro and in vivo varying sensitivity to Ftorafur. Ftorafur does not increase the frequency of SCE.

Inhibitory effect of human urinary extract on the growth of DMBA-induced mammary tumors in Holtzman rat.

The effect of a crude protein fraction from human urine having gonadotropin inhibitory activity (GGIM) was observed on the growth of 7,12-dimethylbenz(a)anthracene induced mammary tumors in Holtzman rat. Results show that while in 12 out 13 tumors, tumor growth was retarded/regressed following 2--4 weeks of CGIM treatment, tumor growth in control rats was unabated. However, no positive correlation with hormone dependency and tumor regression could be established. Further, this crude anti LH material was found to be non-toxic to tumor cells in vitro.

Specific, antigenic protein of Guerin epithelioma.

Specific, antigenic protein of the molecular weight 61 000 and 66 000 and isoelectric point 5.15 were isolated from cytosol of Guerin epithelioma and antiserum against these proteins was obtained. These antigens appear to be specific for epithelioma. Such antigens were not found in any normal rat organ. Blood serum of Guerin epithelioma bearing rats does not contain antibodies against these antigens.

The chemotherapy of C3H mice bearing Gardner lymphosarcoma with bovine fibrinogen-methotrexate derivative.

Derivative of bovine fibrinogen (FBG) containing chemically bound methotrexate (MTX) has been prepared by action of ethyldimethylaminopropyl carbodiimide. The derivative retained its solubility and clotability. Intraperitoneally administered FBG-MTX derivative one day after transplantation of the ascitic Gardner lymphosarcoma prolonged distinctly the survival of C3H mice. The intravenous application of FBG-MTX derivative to mice bearing the solid form of the tumor exerted chemotherapeutic effect resulting in prolongation of survival. The local application of FBG-MTX solutions followed by injection of thrombin resulted in the formation of a fibrin clot in the tumor area which persisted at least 48 hours. Local chemotherapy of the solid tumor with fibrin clot containing MTX performed on day 1 or 3 led to significant prolongation of survival of the treated animals. Mechanism of MTX liberation and the possible application of FBG-MTX derivative in chemotherapy of tumors are discussed.

Changes in hemopoiesis of mice of the C3H strain following transplantation of Gardner lymphosarcoma and infection with LDH-virus. II. Spleen and bone marrow.

Cytological examination of spleens of mice with growing Gardner lymphosarcoma contaminated with LDH virus indicates the augmentation of erythropoiesis and granulopoiesis, as well as the decrease of lymphopoiesis. Similar changes were observed after infection with LDH-virus. The counts from the bone marrow smear showed more pronounced changes only in tumorous mice, in which repeatedly increased granulopoiesis was found. When the mice bearing Gardner lymphosarcoma were treated with L-asparaginase, then the cytological findings in the spleens and marrows of femors were almost normal. The examination of spleen and bone marrow smears revealed no tumor cells. Histologically examined spleens of mice with 12-day-growing Gardner lymphosarcoma demonstrated infiltration of the follicles with tumor cells. In the marrow of sternum no infiltration of tumor cells could be established.

Changes in hemopoiesis of mice of the C3H strain following transplantation of Gardner lymphosarcoma and infection with LDH-virus. III. Blood proteins.

Inoculation of bone marrow and spleen cells of C3H/Sumice strain mice demonstrated that on the ninth day of growth of Gardner lymphosarcoma these tissues were invaded with tumor cells. The weights of mice with advanced tumors increased. Yet the deterioration of health status of mice is characterized by decreased food and water consumption, as well as by lower concentrations of proteins in mouse sera and plasma. The albumin fraction shows a remarkable drop. These changes could not be detected in tumor free mice following LDH-virus infection. The treatment with L-asparaginase influenced the manifestations of tumor growth, but not the effectivity of LDH-virus contaminating the tumor as demonstrated by the increased activity of lactate-dehydrogenase in the mouse sera.

A morphological study of continuously irradiated lymphosarcoma cells.

Mouse lymphosarcoma LS/BL cells, cultivated as a free suspension in the peritoneal cavity of C57Bl mice, were continuously irradiated by 60Co unit at an exposure rate of 35.8 x 10(-5) mC kg-1 s-1 (5 R/h). After 250 and 350 weeks of irradiation these cells were examined in light and electron microscope. Both the cytoplasm and the nucleus underwent morphological alterations under continuous exposure. Areas of advanced cytoplasmic degradation were often observed and many nuclei showed wide indentations and prolongation of their shape. The number of mitochondria and vacuoles in the cytoplasm was increased markedly in the irradiated cells. The authors consider some of the described alterations caused by continuous irradiation to act as compensatory mechanisms of the impaired cell.

Long-term remission in acute lymphoid leukemia patients on maintenance chemoimmunotherapy.

Comparative effectiveness of maintenance chemo- and chemo-immunotherapy with Soviet strain of BCG vaccine was studied on 75 children with acute lymphoid leukemia (ALL). The patients were divided in two groups: I -- 25 patients received maintenance chemotherapy plus BCG; II -- 50 patients were given sole form of maintenance chemotherapy. Median duration of remission in the group I -- 36.3 +/- 4.2 months; in the group II -- 13.8 +/- 1.9 (p less than 0.001); median duration of life -- 45.1 +/- 3.7 and 23.9 +/- 3.3 (p less than 0.001) respectively. In 6 out of 25 patients (group I) the first complete remission continues for more than 6 years (median duration of remission -- 82.5 and life -- 85.3 months). In the group II the first remission continues only in 1 patient out of 50.