Kinetics of 5-aza-2'-deoxycytidine phosphorylation in mouse spleen and L1210 leukemic cell extracts.

Out of different nucleosides and deoxynucleosides testes for their ability to block the phosphorylation of labeled 5-aza-2'-deoxycytidine in the presence of ATP and the cell-free extract from mouse spleen only deoxycytidine and cytosine arabinoside depressed the reaction significantly. With the same system the phosphate donor specificity for 5-aza-2'-deoxycytidine and deoxycytidine was determined. All of the 5'-triphosphates used were less efficient donors with respect to the analogue than to the natural substrate. The apparent Michaelis constants for the phosphorylation of 5-aza-2'-deoxycytidine and deoxycytidine were 2.9 and 2.5 x 10(-5) M, respectively. Using the extract from L1210 leukemic cells the Km constant for 5-aza-2'-deoxycytidine was 6.4 x 10(-5) M and that for deoxycytidine 2.7 x 10(-5) M. The Ki constants with the spleen extract were 1.2 x 10(-3) M for 5-aza-2'-deoxycytidine and 5.8 x 10(-6) M for deoxycytidine. Both compounds acted as competitive inhibitors of one another.

Cell-surface adhesiveness of mouse leukemias.

Cell-surface adhesiveness of forty-one primary mouse leukemias was quantitatively examined by the Latex (polystyrene) particle adherence (LPA) assay. Mean surface adhesiveness of leukemic cell populations was found to be substantially lower than that of normal mouse thymus, lymph node and spleen cell populations. No significant differences were found among mean LPA-positivity of sixteen thymic lymphomas, sixteen reticulum cell sarcomas and six myelogenous leukemias examined; the LPA-positivity of two nonthymic lymphomas and one undifferentiated leukemia was higher and comparable with that of normal mouse thymus cells. Comparison of the LPA-positivity of normal thymus cells and thymic lymphomas indicated that the malignant conversion of thymus cells was either accompanied or followed by a decreased cell-surface adhesiveness.

Demonstration of C-type viruses in N-methyl-N-nitroso urea (MNU)-induced leukemia of mice by reverse transcriptase activity and XC assay.

A significantly higher MuLV expression was demonstrated in cells of MNU-induced leukemia of mice compared to corresponding cells of untreated control animals by reverse transcriptase activity and XC cell assay. These positive findings were verified by determination of indirect immunofluorescence test to look for intracytoplasmic MuLV p30. The problem is discussed whether these viruses play a role in chemical leukemogenesis.

Common antigenic determinants on structural proteins p10--12 of PMFV and MPMV type D retroviruses.

PMFV, a type D retrovirus isolated from a malignant human embryo cell line, was compared with Mason-Pfizer monkey virus (MPMV) in a sensitive tannic acid enhanced indirect immunodiffusion test. In addition to the previously shown common antigens, both viruses contain identical group-specific antigenic determinants on their p 10--12 as demonstrated with a specific p 10--12 MPMV test system. Interspecies mammalian type C virus antigens were not detected in highly concentrated PMFV preparations.

Significance of radioimmunoassay of human chorionic gonadotropin and alpha fetoprotein in nonseminomatous germ cell tumors of the testis.

Radioimmunoassays of human chorionic gonadotropin (HCG) and alpha fetoprotein (AFP) made in 49 patients with nonseminomatous testicular tumors have shown that these investigations make the diagnosis more precise, permit to follow up the dynamics of the course of the disease and the effectiveness of treatment and may help to reveal the presence of otherwise undetectable tumorous metastases. The significance of the these assays is enhanced if the two tumorous proteins are investigated in parallel. The results proved rightly positive in 43 (87.8%) and falsely negative in 6 (12.2%) of the patients. The absence of HCG and AFP production in some of the patients with an active disorder has not as yet been elucidated.

Immunocompetence of splenocytes from mice bearing different-aged tumors assessed in lymphocyte-induced angiogenesis assay.

Inbred Balb/c mice bearing 2- or 4-week-old transplantable syngeneic sarcoma L-1, when injected intravenously with the cells of the same tumor, displayed a low or high number of tumor nodules in the lungs, two weeks after the injection. In order to test whether this phenomenon is dependent on changes in host immunity associated with tumor progression, the immunocompetence of spleen cells from tumor-bearing mice was measured in a lymphocyte-induced angiogenesis assay, performed on (C57Bl/Sn X Balb/c)F1 recipients. Splenocytes from mice bearing tumors of different age, whether treated or not with cyclophosphamide, showed, however, the same angiogeneic potency.

Diagnosis of malignant tumors on the basis of the current generating capacity of malignant tumorous tissue. I. Examination in vitro, animal experiments and endoscopic measurements.

On the basis of the different electrolyte composition and lactic acid content of malignant tumorous tissue an electrochemical measuring method was worked out for the quick-diagnosis of malignant tumors. The in vitro model experiments and the measurings carried out on mice with Crocker's sarcoma proved the practicability of the electrochemical technique. The present study contains the measuring results of 55 adenocarcinomas and 12 scirrhous carcinomas. The current generating capacity of adenocarcinomas -- both with endoscopic measuring and that performed of samples excised -- exceeds the values measured in the healthy surrounding tissue with 45 to 50%. On the other hand, the generating capacity of scirrhous carcinoma is with 50% lower as the value measured in the healthy tissue. The generating capacity of gastric ulcer and polyp is virtually identical with that of the healthy gastric mucosa. The difference in generating capacity between the healthy tissue and malignant tumorous tissue is statistically significant.

Characterization of tumors induced in adult Macaca mulata monkeys with Carr-Zilber strain of Rous sarcoma virus.

The Carr-Zilber strain of Rous sarcoma virus is highly oncogenic for adult monkeys of the species Macaca mulata. In the present study, we examined some characteristics of tumor growth when the tumor either regressed or progressed until it killed its host. We followed the kinetics of antibody formation to antigens coded by CZ-RSV in animals with a different course of tumor disease. The sera of animals tested contained virus-neutralizing antibodies, in addition to gs antibodies. The results are discussed with regard to the possible virus-productive type of interaction between RSV and the monkey cell.

Neoplastic growths in chickens treated with cell and cell-free material from transplantable hepatoma induced by virus strain MC-29.

Series of experiments were carried out with chickens (line 15-I and Canadian white leghorn) for establishing the oncogenic properties of Mc-29 virus strain after its multiple replication in cells transformed by it (hepatoma cells). It was established that: 1. After s. c. and i. m. inoculation of intact hepatoma cells local hepatomas of varying size developed after 8--15 days accompanied with general viremia; after i. p. or i. v. injection multiple hepatoma growths occurred in the visceral organs and bone marrow. 2. The i. v. or i. p. inoculation of cell-free material of hepatoma or blood plasma of hepatoma bearing chicken lead after 48--80 days to the formation of hemocytoblastic and myelocytoma growths in the visceral organs and in the bone marrow and exclusively rare--of primary liver and kidney carcinomas. After s. c. injection of cell-free material the result is negative. A conclusion could be drawn that strain Mc-29 virus though replicated in the transformed hepatoma cells preserved its basic leukemogenic potentialities to induce myelocytomatosis and hemocytoblastosis and very rarely--liver and kidney carcinomas.

Nephroblastoma and renal mesenchymal tumor induced in rats by N-nitrosoethyl- and N-nitrosomethylurea.

Rats treated prenatally with N-nitrosoethylurea or with N-nitrosomethylurea developed nephroblastomas, renal mesenchymal tumors and epithelial tumors (adenomas and carcinomas) of the kidneys. 15 nephroblastomas and 59 mesenchymal tumors were examined histologically. Nephroblastomas were encapsulated growths composed of dark cells, forming primitive tubules similar to those seen in the rat embryonal kidney and in human Wilms' tumor. Mesenchymal renal tumors showed an infiltrative growth and consisted of fibroblast-like cells, smooth muscles and angiomatous areas with the engulfed pre-existing tubules. These growths are similar to the mesenchymal renal tumors induced in the rat by N-nitrosodimethylamine. Nephroblastoma and mesenchymal renal tumor are considered to be separated entities, the first corresponsing to the epithelial variant of human Wilms' tumor and the second to congenital mesoblastic nephroma.

Provirus DNA sequences in Rous sarcoma cell genome.

Non-producer hamster and virus-producing chicken Rous sarcoma cells contain a complete avian sarcoma virus (ASV)-provirus DNA (pro-DNA). Unlike this, in normal hamster liver DNA to ASV--specific sequences are absent. Moreover, the nuclear chicken Rous sarcoma and chick embryo cell (CEC) DNA preparations contain practically all endogenous chicken virus (ECV)-specific sequences, while the hamster tumor DNA was annealed with only more than half of the ECV-RNA sequences. Thus, the pro-DNA of permissive hosts contains so-called "sarcoma-specific", "common" and "endogenous--specific" nucleotide sequences. The "sarcoma-specific" and "common" sequences are present in the hamster sarcoma pro-DNA, only. Both the pro-DNA of permissive cells and the pro-DNA of non-permissive cells consist of moderately reiterated and unique sequences. The "common" regions of pro-DNA are localized, mainly, in the unique zone, while "sarcoma-specific" and "endogenous-specific" sequences of pro-DNA - in the moderately reiterated and unique zones. The pro-DNA organization of permissive and non-permissive hosts is discussed.

Radioimmunoassay for major internal structural protein (p21) of the bovine leukemia virus; antibodies detection and viruses identification.

The major internal protein of bovine leukemia virus (BLV p24) was isolated using ion exchange chromatography on phosphocellulose and gel filtration. The specificity of the BLV p24 isolated was checked by both the radioimmunoprecipitation (RIP) and the competitive radioimmunoassay (RIA). No cross reactivity between BLV p25 and the mammalian viruses of type C and D and the retrovirus isolated from human myeloma cells RPMI8226 [8, 17] was detected. The analysis of 429 leukemia-suspected bovine blood sera resulted in the detection of 165 (38.5%) positive and 264 (61.5%) negative blood sera. A correlation of the results of radioimmunoprecipitation reaction of major internal protein p24 and immunodiffusion test on the glycoprotein antigen of BLV was observed.

Comparison of radioimmunoassay for internal protein of bovine leukemia virus with neutralization test employing VSV-BLV pseudotype.

Two methods for the detection of antibodies to the bovine leukemia virus (BLV) in infected animals were compared for their suitability for the early diagnosis of bovine leukemia - pseudotype neutralization test (PNT) employing vesicular stomatitis virus - bovine leukemia virus pseudotypes (VSV-BLV), and radioimmunoassay test (RIA) for major internal viral protein p24 of the BLV. The comparison was made using more than 300 sera from cows of the herds with high incidence of bovine leukemia. In infected animals the presence of antibodies against virus envelope glycoprotein detected by PNT and antibodies against major structural viral protein p24 detected by RIA were found always coincidentally. Both methods were found highly comparable and suitable for early detection of bovine leukemia virus infected animals.

Karyological and isoenzyme characterization of established human sarcoma cell lines.

Six established human sarcoma cell lines (giant cell tumor of bone B-5GT, fibrosarcoma, B-6FS, cystosarcoma phylloides B-19CS, synovial sarcoma U-4SS and two osteogenic sarcomas U-20S and U-393OS) have been studied and compared to the normal B-41FB fibroblastic cells and the HeLa cells. For cytogenetic and isoenzyme characterization both conventional and G banding techniques as well as the mobility of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PD) were employed. All six sarcoma cell lines had karyotype and LDH patterns of human cells. A study of the chromosome counts distribution revealed a large variability from one sarcoma cell line to the other. There was no evidence of any cross-contamination between the lines or by HeLa cells. This conclusion is based on the detection of G-6-PD with B phenotype, the lack of HeLa marker chromosomes in all sarcoma lines and the presence of Y chromosome in U-4SS and U-393OS derived from male donors. In addition, each sarcoma cell line revealed a group of distinctive marker chromosomes which can serve to identify them and help to control cell line specificity during in vitro culturing.

Inhibition of polyoma virus-specific RNA formation by 5-bromo-2'-deoxyuridine.

The synthesis of polyoma (Py) virus-specific RNA, assayed by molecular hybridization at late phase of infection of mouse embryo (ME) cells is inhibited by 5-bromo-2'-deoxyuridine (BrdUrd). 0.25-12.68 microgram/ml BrdUrd causes an inhibition to about 60-10% of the control, respectively.

Isolates of avian sarcoma virus Bratislava 77 (B77) oncogenic for mammals do not contain an excess of transformation-defective mutants.

Several isolates of the avian sarcoma virus Bratislava 77 (B77), used in tumor induction in rats, hamsters and mice, were tested for the excess of spontaneously segregated transformation-defective mutants (tdB77). The question was asked whether these td mutants could interfere with transforming sarcoma viruses at tumor induction in mammals. It was found that the B77 virus isolates used in successful sarcoma induction in mammals did not contain an excess of td mutants. One virus isolate which had an excess of td mutants did not induce tumors in mammals. The further characterization of the rescued viruses from virogenic mammalian cells showed that all rescued viruses had the same sub-group C specificity as the original isolate of B77 virus. The integration of the viral genome into mammalian cellular genome did not alter transforming ability of the rescued viruses on duck embryo cells. It seems that propagation of B77 virus in conditions in vivo did not support the segregation, and accumulation of an large excess of td mutants in stocks of B77 virus.

Glycoprotein and protein composition of bovine leukemia virus (BLV).

Highly purified preparation of bovine leukemia virus (BLV) was obtained by the method described in this communication. BLV was isolated from cell-free culture medium, harvested from BLV-producing cell-line FLK. Polyacrylamide gel electrophoresis of BLV proteins and glycoproteins, in comparison with avian myeloblastosis virus (AMV), confirmed high purity of BLV preparations. Molecular weights of some BLV-proteins and glycoproteins were calculated. By employing polyacrylamide gradient 5 to 30% it was found that BLV structural protein p15 contains at least two components.

Proliferation rate--drug sensitivity relation in clones isolated from heterogeneic tumor cell population.

Four separate cell clones were isolated from parental rat RBB/R cell line. Sensitivity of clones to 3-oxauracil varied, and was dependent on the proliferation rate of particular clone. Colony formation method was more appropriate than 3H-thymidine incorporation for determination of post-treatment surviving cells.

Varicella-zoster antibodies in patients with Hodgkin's disease.

Statistically significant differences in mean varicella-zoster antibody titre was found between the normal population and Hodgkin's disease group. No difference in mean antibody titre between the four histological categories of the disease was found.

Alterations in erythrocyte enzymes in cancer.

An increase in lactate production, and in activities of phosphohexoisomerase, phosphofructokinase and pyruvate kinase was found in erythrocytes of patients with advanced cancer disease. Phosphofructokinase isolated from patients' erythrocytes showed enhanced affinity for substrate and coenzyme, diminished thermal stability and changed dependence of the activity on pH. Allosteric properties of the enzyme were modified. A decrease in glucose-6-phosphate dehydrogenase activity was observed in hemolysate. The partially purified enzyme showed decreased affinity for glucose-6-phosphate, and markedly reduced stability at 45 degrees C and in the acid and alkaline pH range. Changes in kinetic and molecular properties of the two key enzymes of glucose metabolism may contribute to hemolysis observed in many cancer patients. Incubation in vitro of normal human erythrocytes with blood sera of patients resulted in increasing of phosphofructokinase, and decreasing of glucose-6-phosphate dehydrogenase activity, indicating that an acquired enzymopathy may be present in cancer.

epsilon-N-trimethyl-lysine: a natural cell component with mitogenic activity.

The blastogenic capacity of a new mitogenic agent, epsilon-N-trimethyl-lysine (TML) was examined on human peripheral blood lymphocytes. The changes in relative DNA and histone content after TML treatment testified that activated lymphocytes passed through the whole cell cycle. The comparison of the 3H-TdR incorporation rate into unseparated T-B lymphocyte and purified T lymphocyte cultures suggested a preferential effect on the thymidine incorporation of T lymphocytes. TML induced preferentially high responses in cases of blood cells taken from women. The possibility of DNA repair mechanism could be excluded. The results of combined TML-PHA treatment experiments suggested a difference in the trigger mechanism of these two mitogenic compounds.

Chemical structure--acute toxicity relation of m-substituted derivatives N,N-dimethyl-4-aminoazobenzene with hepatocarcinogen effects.

The mol LD50 values were correlated with Hammett sigma constants in terms of the Hansen empiric equation 7 for meta substituted derivatives of N,N-dimethyl-4-aminoazobenzene. Good agreement and the line slope of the correlation was achieved. Relation acute toxicity--carcinogenity was discussed in the end.

Immunological determination of tyrosinase in sera of melanoma-bearing hamsters.

The quantitative ring immunodiffusion technique was adapted for the determination of the DOPA-oxidase activity of tyrosinase [E.C.1.14.18.1] in the sera of melanotic melanoma-bearing hamsters. The smallest amount of tyrosinase detectable in antibody-agar plates was about 0.5 micrograms/ml. The quantity of tyrosinase in the hamster sera depended on the grade of tumor development and rose to a value of 40 microgram/ml. There was no detectable activity in the sera of hamsters without tumor. The possibility of using this method to test sera of patients and horses with melanoma malignum is discussed.

Immunological reactivity in the lymph nodes of the host following experimental cancer immunotherapy utilizing polymerized autologous tumor tissue particles.

The effects of the specific active cancer immunotherapy utilizing autologous tumor tissue particles polymerized with ethylchlorformiate on the immune system of the host were evaluated in DBA/2 mice bearing a malignant mast cell tumor, mastocytoma (P-815 X 2), with the special emphasis placed on the morphologic changes in the lymph nodes. In assessing the lymph node morphology in relation to the immunologically reactive lymphocyte populations (T- and B-cells), the standardized reporting system was employed, and the post-capillary venule score (PCV-S), shown to be related to the T-cell activity, was calculated for each lymph node. The specific cancer immunotherapy instituted along with the PPD tuberculin as adjuvant was capable of reverting to a considerable degree the profound depletion of the T-cell population in the paracortex of the lymph nodes, as well as exerting a stimulatory influence on the B-cells responsible for antibody synthesis in the cortex and medulla of the nodes. The results were interpreted to favor the view that the favorable influence on the tumor rejection previously shown to be exerted by the specific immunotherapy technique studied, most probably is attributable to the observed stimulatory effects of it on the lymphocyte populations involved in cell-mediated and humoral immune responses. The appropriate cooperation of both T- and B-lymphocytes is most probably needed to ensure the most effective host response against the tumor cells in this system.

Individual serum proteins and acute phase reactants in monoclonal immunoglobulinopathies. A study in patients with macroglobulinemia.

Thirty-four patients with primary macroglobulinemia (Waldenström) were compared with healthy subjects and myeloma patients previously studied regarding their levels of 17 specific serum proteins. Quantitative changes of individual serum proteins were discussed with the respect to their biologic functions. Moreover linear correlations between the serum proteins studied were examined. Selected hematological data at diagnosis of macroglobulinemia have been also reported. The concentration of IgM monoclonal component (MC) was in 88% of macroglobulinemic sera above 1000 mg/dl. If no hyperproteinemia and IgM excess are concerned 5 proteins were found elevated (orosomucoid, alpha1-antitrypsin, ceruloplasmin, C-reactive protein (CRP), hemopexin) and 7 were decreased (prealbumin, albumin, alpha2HS glycoprotein, transferrin, C3-component, IgA, IgD). Differences in mean levels of 4 proteins (haptoglobin, alpha2 macroglobulin, beta2-glycoprotein I, IgG) did not reach statistical significance. CRO was demonstrable in two-thirds of patients and the CRO levels more than 1 mg/dl were noticed in a half of them. changes of phase proteins in macroglobulinemia were similar to those observed in myeloma and other malignancies. In the present series as previously in myeloma no positive relationship could be demonstrated between the concentration of MC and individual acute phase proteins. The levels of polyclonal IgG and IgA, not IgD were found significantly higher in macroglobulinemia than in myeloma, thus polyclonal formula in macroglobulinemia being more favorable. In contrast to myeloma the IgM MC showed at 5% level no significant negative correlation with polyclonal immunoglobulins (IgG, IgA, IgD) among which positive mutual correlations were noticed.

On some similarity between membrane antigens of the cell of Zajdela hepatoma and liver of rats subjected to a single 4-dimethylaminoazobenzene injection.

Zajdela ascitic hepatoma cells were tested with the organ specific immune serum against kidney cell ghosts in the reaction of immunofluorescence. On the surface of the hepatoma cells occurred heteroorganic antigens characteristic of the membranes of intact rat kidney but never observed in their liver. Similar antigens were found in the liver of rats 1-18 days following a single injection of a hepato-carcinogen--4-dimethylaminoazobenzene but not after 4-diethylaminoazobenzene, which is a noncarcinogenic structural analogue of 4-dimethylaminoazobenzene. Noteworthy is the transient nature of this alteration in antigenic structure, since from day 21 after carcinogen application it was no longer found.

Combined therapy of L1210 leukemia with cyclophosphamide and lycurim.

The authors treated DBA/2 mice with L1210 leukemia with cyclophosphamide (CY) plus lycurim (LY), both alkylating agents. There was established a dose-dependent antitumor effect after early single treatment with CY and LY. Sequentional combined treatment with CY and LY induced an enhancement or synergism in therapeutic effect of drugs, more related to the doses used than to the sequence of drug administration. The optimal synergistic effect, as well as "cure" of treated animals were observed when both drugs were used in doses close to LD10.

Serum xanthine oxidase in breast carcinoma.

This study comprises 46 patients with various stages of breast carcinoma and 49 normal women as control group. Serum xanthine oxidase and uric acid levels were determined. Assessment in terms of changes of the enzyme xanthine oxidase was carried out prior and after surgery as a short term follow up. There was a significant fall of serum xanthine oxidase in patients with breast carcinoma. The level of serum xanthine oxidase was found to be varied with the stage of cancer.

Bone sarcomas in 239Pu-treated mice.

Female ICR mice were injected with a monomeric form of 239Pu (180.1 kBq/kg) at the age of 10 weeks and during the long-term contamination the incidence of bone sarcomas was studied. Average survival of 100 control mice was 545 +/- 14 days, the 100 plutonium-given mice survived 420 +/- 10 d and 50 sarcoma-bearing animals 456 +/- 11 days. Most of the 61 induced sarcomas were located in the spine (70%) and in trabecular parts of the other bones. Among these 61 tumors there were 55 osteoblastic osteosarcomas, 4 immature undifferentiated sarcomas and 2 angiosarcomas. In 6 tumor-bearing mice metastases were observed in lungs, liver, intracranial cavity and in mediastinal lymph nodes. Among control mice only one osteogenic mandibular sarcoma was verified. The osteosarcoma incidence was evaluated with respect to the retained activity and cumulative average skeletal dose. The results were discussed and compared with data published by others.

Immunological approach to the treatment of acute lymphoblastic leukemia.

On the basis of experimental results an immunological method of acute lymphoblastic leukemia treatment by means of specific immunity regulation was designed. The administration of antibodies (immunoglobulins) with prednisolone to patients induced 100% remissions in a comparatively short time. None of the patients treated with immunoprednisolone therapy had neuroleukemia. The immunological findings indicate that the therapeutic effect depends on the "deblocking" action of the applied immune preparation.