Potential anticancer agents. XXII. Pharmacological properties of some new triazene derivatives.

The pharmacological properties (toxicity and antitumor activity against W256 carcinosarcoma and L1210 leukemia) of thirty five new triazenes derived both from amides and esters of 3-methyl-4-amino-benzoic acid and from aromatic dipeptidic esters were investigated. The new compounds proved to be moderately effective against W256 carcinosarcoma and totally devoid of activity against L1210 leukemia, except for ethyl,4-(3,3-dimethyltriazenophenyl)-N-alpha-(9-fluorenyl)-acetyl-L-alanine ester which exhibited borderline activity against this leukemia. The pharmacological data thus obtained were correlated with the physico-chemical parameters of the new triazenes in a quantitative structure-activity relationship (QSAR approach). The results of the computed equations suggested that the electronic and the lipophilicity factors alone are not sufficient to give a satisfactory picture of the in vivo behavior of such compounds.

Contact of migrating L5222 leukemia cells with the substrate as studied under the action of gravitational force.

The interaction of actively migrating L5222 cells with the serum-coated glass surface was studied under the influence of an external force. In order to generate such force, the plane on which the cells were migrating, was inclined at different angles. The analysis of the L5222 cell migration pattern reveals that the modification of the cell movement direction relative to the surface appears when the plane is inclined at 1 degree. The increase of the slope to 2 degrees makes this effect more evident. When the slope is equal to 3 degrees the active motion of L5222 cells seems to disappear. The component of the gravitational force parallel to the plane on which the cells migrate ranges from 6.3 X 10(-15) N to 1.9 X 10(-14) N for the slopes from 1 degree to 3 degrees, respectively. The results of the study indicate that the force of interaction of migrating L5222 cells with the substrate is very small in comparison with the force of adhesion (not accompanied by locomotion) and seems to be comparable to that which operates in the near-substrate region of non-locomotive cells (e. g. L1210). Thus, the cell-substrate contact related to active movement and the non-locomotory adhesion of L5222 cells are probably different phenomena. As only L5222 cells were studied in this work, the above conclusions should be restricted to this cell type only.

Recovery of hematopoiesis in bone marrow of mice after continuous irradiation with dose rate 4.8 Gy/day.

In this paper the morphological changes in mouse bone marrow till day 60 after the termination of continuous irradiation with the dose rate 4.8 Gy/day (480 rad/day) and the total accumulated doses 9.6 Gy (960 rad) and 19.2 Gy (1920 rad) are evaluated. Many mononuclear cells of the morphologically lymphoid type appear in the bone marrow on day 9 of the recovery and later on extensive granulopoiesis recovery occurs reaching the control level, as early as on day 21--28 after the irradiation. The recovery of erythropoiesis and megakaryocytopoiesis proceeds more slowly and their total recovery occurs within the period of 28--60 days after the irradiation. The total accumulated dose 19.2 Gy (1920 rad) causes irreversible damage in the bone marrow and the death of all experimental animals occurs within 11 days after irradiation.

The identification of Thormählen positive melanogen "A" from the urine of melanoma patients as 6-methoxy-5-indolylglucosiduronate.

The Thormählen positive melanogen previously determined as "A" has been isolated from melanotic urine by means of column chromatography on Amberlite IR 45, DEAE cellulose and Dowex 50. According to its behavior during a study with 1H--NMR spectra it has been found that the isolatd compound is 6-methoxy-5-indolyglucosiduronate, the metabolite of 5,6-dihydroxyindole. The possible formation and biotransformation of this compound is discussed.

Colony formation in agarose gels containing 2-deoxyglucose: its relationship with malignant transfusion.

At the light of the increasing evidence that malignant transformation is a multistep process the wide correlation between growth in soft agar and various degrees of tumorigenicity suggests that this could be a property of the early stages of malignant transformation. In the present paper we give evidence indicating that colony formation in agarose gels containing deoxyglucose can operate as a more restrictive criterium than growth in soft agar for malignant transformation in vitro, possibly associated to advanced stages in this process.

The Eker renal tumor rat. Tumor transplantability and a re-evaluation of tumor histology.

Further histological findings for the naturally occurring kidney tumor of the Eker rat included the unusual location of the tumor entirely within the medulla rather than in the more usual site within the kidney cortex; the presence of psammoma bodies, and metastases of the tumor in one animal. The kidney tumors were shown to be transplantable with a latent period of about nine months. Histologically, the transplanted tumor, even at the twenty-eight transplant generation, maintained the cellular characteristics of the primary. The histological and transplant data allow the classification of the definitive tumor as a primary adenocarcinoma. The suitability of the tumor as an animal model for human renal carcinoma is suggested.

Acute phase reactants and clinical stages in multiple myeloma.

Serum levels of seven specific proteins mostly acute phase reactants (APR) have been studied (transferrin, alpha 2-macroglobulin, alpha 1-antitrypsin, haptoglobin, C3, ceruloplasmin, orosomucoid) in 14 healthy subjects and in 55 patients with multiple myeloma. The alpha 2-macroglobulin and transferrin are significantly decreased in the myeloma group compared with healthy controls whereas the remaining proteins under study are elevated, significantly only orosomucoid and ceruloplasmin. The APR in the IgG3 myelomas seem to show differences from those of the IgG1 and IgG2 subclasses the levels of the negatively changed proteins being lower, whereas the positive APR higher in the IgG3 myeloma. In the IgA myeloma, however, decreased levels of haptoglobin and alpha 1-antitrypsin have been found in spite of being positive APR. Transferrin and haptoglobin serum level can be included as a new parameter in regression equations for calculation of IgA myeloma cell mass.

Alterations of plasma levels in malignant lymphoma.

Fibrinogen levels were investigated in the plasma of a group of patients with malignant lymphoma consisting of M. Hodgkin's and non-Hodgkin's lymphoma, and were found to be significantly higher in an acute onset of the disease and in relapses, than in a control group of healthy blood donors. In the terminal stages, fibrinogen levels in the plasma were observed to decline towards the upper normal limits. Plasma fibrinogen levels are considered to be a good indicator of the activity of the disease.

Hormonal therapy and alterations of immunity in prostatic cancer.

Retrospective evaluation of cell-mediated tumor-associated immunity in patients with prostatic cancer before and after the receipt of non hormonal or hormonal therapy disclosed that patients receiving hormonal therapy, i. e., orchiectomy and/or oestrogen, possessed significantly lower levels. The potential clinical relevancy of the suggested immunosuppressive effects of hormonal therapy on tumor-host responsiveness is considered in view of the present and previous observations.

Treatment of ectopium with low-output He-Ne laser.

The stimulatory effect of laser on the physiological healing process of the portio surface has been studied. It has been shown that He-Ne laser, 632.8 nm, 5 mV, 1 J/cm2, has a beneficial effect. 85 patients out of 100 recovered, 10 improved, 5 patients were left out of evaluation. No pathological changes were found at colposcopic and cytological controls.

Ambient air pollution and lung cancer incidence in 1965--1975 among native population of an industrial city.

Lung cancer incidence in Zabrze (Upper Silesian Region, Poland) in the years 1965--1975 among native population was analyzed in connection with industrialization level and air pollution. In comparison with other towns or rural counties of Poland, the native population of the industrial city Zabrze is characterized by a lower risk of lung cancer in spite of the fact that the native population of the city has been exposed for a long time to different harmful chemical impurities of air.

Computation of cancer incidence rates for defined small geographical areas--matching of numerator and denominator.

The authors present the problem of validity of numerator and pertinence of denominator for small geographical areas on sample of average annual crude incidence rates of stomach cancer in Slovenia during the period 1968--1974. At census in 1971 this country counted 1 727 137 of population. According to the residence of the patients the rates have been calculated for 60 municipalities, and besides for 29 so called "epidemiological regions", defined by their geographical and ethnic characteristics. Although in both instances great differences in incidence rates have been found, only a few areal units exhibited a significantly lower or higher rate from the national average. Specially areas with small population base "disappeared" in the average, although the value of the rate for them was rather low or high. A formula is presented by which at given rate for total country it is possible to determine the minimal size of population which still could provide estimation of statistically significant lower rate. Thus, for areas with small population a period of observation through many years is required in order to obtain the necessary minimal population base. The question is pointed out, whether this approach is reasonable as to the epidemiological interest, considering that some factors related to cancer may change during the long period of time. On the other hand, when merging small areas into larger ones, attention must be paid that these are not too heterogeneous as to their cancer incidence rate, geographical, ethnic, socio-economic and life-style features.

Action of misonidazole on L5178Y-R and L5178Y-S cells. I. Comparison with antimycin A and potassium cyanide under aerobic conditions.

Effects of treatment with misonidazole under aerobic conditions were examined in L5178Y-R and L5178Y-S cells. These cells slightly differ in susceptibility to the drug, however, the effects of treatment are similar: 1. slowing down growth without affecting viability at low doses; 2. cell lethality at higher doses with rapid elimination of dead cells from the population and resuming normal growth; 3. decrease in 3H-thymidine incorporation. These effects of misonidazole were strikingly similar to those of antimycin A (1 microgram/ml) and KCN (1 mM). It seems that the common cause of the described manifestations of cellular damage inflicted by all three chemicals is energy depletion due to disturbances in the function of mitochondrial electron transport.

Action of misonidazole on L5178Y-R and L5178Y-S cells. II. Cytotoxicity and radiosensitization under hypoxic conditions.

Misonizadole exerts an equal cytotoxic effect on L5178Y-R and L5178Y-S cells; 5 mM misonidazole sensitizes cells of both strains to a similar extent under hypoxic conditions: the effect of drug in 59% of the oxygen effect in L5178Y-R cells and 52% in L5178Y-S cells. It was previously reported that the amount of initial DNA damage determined by the alkaline unwinding technique is 1.3 times lower in irradiated hypoxic L5178Y-R cells than in L5178Y-S cells. Hence it seems that the drug ability to sensitize these cell strains does not depend on amount of initial DNA damage available for interaction. Although the examined L5178Y strains differ in radiosensitivity, their ability to repair oxygen and misonidazole-dependent cellular damage is similar.

Cytogenetic study of malignant and benign effusions.

The direct chromosome study of mitotic cells in effusions was performed in 49 patients with malignant and 5 with benign effusions. Cytogenetic analysis correctly identified 69.3% of malignant effusions, standard cytology 79.6%. With both methods combined 95.9% of all cases were correctly diagnosed as malignant. The presence of mitoses in the effusions was found to be an unfavorable prognostic sign and was correlated with a shorter survival. Chromosome analysis of the malignant effusions revealed stemlines with extensive numerical and structural rearrangements. No consistent marker chromosome was detected in the histologically similar tumor types. Normal chromosomes. No 1 and 14 were preferentially lost from the karyotype of malignant cells. In two samples double minutes were observed.

Clinical usefulness of serum acute-phase reactants in patients with ovarian tumors.

Concentrations of the following acute-phase reactants (APR) were determined in the sera of patients with benign and malignant ovarian tumors: haptoglobin, ceruloplasmin, sialic acid, seromucoid (in sulphosalicylic acid supernatant), and trypsin inhibitory capacity. In patients with ovarian carcinomas significant increase (p less than 0.001) in all measured APR parameters as compared to healthy women and women with benign tumors was found. Moreover, the levels of haptoglobin, ceruloplasmin and trypsin inhibitory capacity were statistically lower (p less than 0.001) in nonmalignant than in malignant ovarian disease. No distinct statistical difference was confirmed between healthy controls and the benign tumor group as well as between ovarian carcinomas of Stages I--III and Stage IV.

Condylomatous lesions associated with precancerous changes and carcinomas of the uterine cervix.

The present communication is a comparative survey of the histological specimens derived from the dysplastic or neoplastic lesion of the uterine cervix of 400 women comprising two age-matched series (200 women in each) collected from two Finnish hospitals. Special emphasis was placed on the detection of the newly described condylomatous lesions (flat, inverted, papillomatous) and the possible variations in their biological behavior in the two series. The biological behavior of the condylomatous lesions (the distribution into the three types, their high frequency in young women, and their relationship to different degrees of epithelial atypia) was not significantly different in the two series studied. The main difference between the series was in the frequency of the condylomatous lesions which was markedly higher (p less than 0.005) in the material made up of women in eastern Finland. The results suggest that condylomatous lesions caused by human papilloma virus (HPV) are ubiquitous and characterized by a rather constant biological behavior. The reasons behind the observed local variations in their frequency remain obscure and advocate an epidemiological study combined with virological assays to be done among the patients with precancerous lesions of the uterine cervix to gain further data on the role of HPV in human squamous cell carcinogenesis.

Nucleolar silver stained granules in rat Yoshida sarcoma cells after RNA synthesis inhibition.

The number of nucleolar silver stained granules representing nucleolus organizer regions in interphase nuclei was studied in Yoshida ascitic sarcoma cells without and after the inhibition of the nucleolar RNA synthesis with actinomycin D or cyclophosphamide. The results demonstrated that the number of nucleolar silver stained granules decreased after the inhibition of the nucleolar RNA synthesis with these drugs disregarding the mode of their action. In addition, the decrease of nucleolar silver stained granules in number produced by actinomycin D was dose dependent. Similarly, the number of nucleoli without silver stained granules increased depending on the dose of the administered actinomycin D.

Electrophoretic mobility profiles of mouse leukemias: I. Electrophoresis of normal mouse thymus and lymph node cell populations.

Anodic electrophoretic mobility of thymocytes and lymph node cells from random-bred ICR Swiss mice was examined. Three cell populations were identified which differed in their surface charge as reflected in their distinct mean AEM and in their time of appearance during the course of life. The first electrophoretically slow-moving population with a mean AEM of 0.97 microns sec-1 V-1 cm was detected in the early postnatal period. In the first month after birth, the first population was replaced by a second, slower-moving population with a mean AEM of 0.83 microns sec-1 V-1 cm. In the second month after birth, a third, fast-moving population with a mean AEM value of 1.24 microns sec-1 V-1 cm appeared in addition to the second cell population. The third population constituted only a minority of thymus cells and continued to be present, as well as the second cell population, up to two years of age. Lymph node cells of ICR mice showed a typical bimodal electrophoretic distribution; fast LNC displayed a mean AEM of 1.18 microns sec-1 V-1 cm whereas slow LNC that of 0.76 microns sec-1 V-1 cm. The percentage of cells in the fast- and slow-moving LNC subpopulations was similar to the expected percentage of T and B LNC. Fractionation of the ICR LNC on nylon wool columns revealed that the majority of the fast-moving LNC corresponded to the nonadherent Thy 1.2+ lymphocytes and the majority of the slow-moving LNC to the nylon wool-adherent sIg+ lymphocytes.

Effectivity of living and non-living BCG vaccine on experimental metastatic spread in mice and the stimulation of the reticulohistiocytic system (RHS).

Heat-killed or formalin-killed BCG vaccine caused statistically significant increases in weight of lungs, spleen and liver, which were in the same range as after administration of equal doses of viable BCG vaccine. Similarly, there was no quantitative or temporal difference in the phosphatase-positive proliferations of the RHS in liver spleen and lungs when using identical doses of viable or heat- or formalin-killed BCG vaccines. The metastasis-prophylactic effect demonstrated for viable BCG was present also after administration of the killed vaccines to a statistically significant degree. The increases in organ weight, extent of phosphatase-positive proliferation foci in liver, spleen and lungs as well as the metastasis-prophylactic effect were entirely identical; they seem to be in a close relationship with each other.

Carcinogenicity of trichloromethine hydrochloride (TS-160 Spofa) and morphological damage after its intraamniotic injection.

The cytostatic TS-160 (trichloromethine hydrochloride, tris-/2-chloroethyl/amine hydrochloride) was injected subcutaneously into SPF Wistar rats of both sexes. After all doses used, spindle-cell or even polymorphocellular sarcomas developes at the injection sites in both sexes: after the dose of 0.1 mg/kg daily for 6 months, in 70%; after 0.25 mg/kg, in 79%; and after 1.0 mg/kg weekly for 6 months, in 45% of the rats. Besides, in 21% of the rats receiving the dosage of 0.25 mg/kg mucus-secreting intestinal adenocarcinomas were found. A relationship was apparent between the incidence figures of subcutaneous tumors on the one hand and the daily injection of the low dose versus the intermittent injection of the dose higher by a decimal order on the other hand. In a concurrent test, TS-160 was given to rats in intraamniotic injection on day 18 of pregnancy in doses of 1, 2.5, and 5 micrograms per embryo. On the first postnatal days, in the affected young rats there were observed morphogenetic disturbances: splanchnocrania and juvenile hair alopecia. These changes became repaired in the further course of postnatal development.

Human malignant melanoma heterotransplanted to nude mice.

Five different human malignant melanoma were heterotransplanted subcutaneously to nude mice. When small tissue pieces were used 3 out of 5 tumors grew. Subcutaneous injections of suspended tumor cells were also made, but all failed to take. Metastatic or infiltrative growth was never seen in the mice observed for up to 2.5 months. The successful grafts largely retained the original morphologicaL features. The three successfully transplanted tumors could all be serially transferred with 100% tumor take. In one case passage time was reduced from 40 days to 15 days. As measured with 3H-thymidine incorporation the proliferation rate increased during the passages. These changes might be due to a selection of more rapidly growing tumor cells in the nudes.

Neutrophil alkaline phosphatase activity in patients with neoplasma during surgical treatment.

In 22 patients with alimentary tract neoplasma, granulocyte alkaline phosphatase activity was determined using the Kaplow's histochemical method. The enzyme activity was determined in peripheral blood before surgical intervention, in blood from vessel draining of tumor before its excision and in peripheral blood 2--3 weeks after excision of tumor. In paralleled tests the enzyme activity was estimated in peripheral blood of 22 healthy individuals. The study indicates that alkaline phosphatase activity of granulocytes is evidently lower in comparison to the controls. Decrease of this enzyme activity in granulocytes from vessels draining tumor and increase of this activity after removal of the tumor suggest that tumor may affect the activity of this enzyme. It seems possible that for decrease of bactericidal capacity of granulocytes in patients with neoplasma the reduction of alkaline phosphatase activity of granulocytes may partly be responsible.

Results of radiotherapy of carcinoma laryngis in relation to the mode of dose fractionation.

The effectiveness of a new fractionation scheme of radiotherapy of carcinoma laryngis was investigated. The irradiation was applied every second day 3 times a week with a fractionation dose of 4 Gy and 3.5 Gy--TD 52 or 52.5 Gy. The results from 95 patients were compared with those from 129 patients irradiated with the conventional fractionation. The cumulative results of primarily healed patients treated with the new fractionation scheme proved significantly better. In supraglottic carcinoma the results of primary healing were on the whole better, although statistical significance could be proved only in stages I and II. In glottic carcinoma, a difference of statistical significance was found in the total number of patients in stages I and III. A higher effectiveness was noted in the 3.5 Gy as against the 4 Gy fractionation. A higher frequency of stronger mucous reactions and delayed radiation oedema occurred with the new fractionation scheme. No complications of a serious degree occurred.

Detection of early forms of breast cancer by mass screening examinations.

Data are presented on the results of the complex prophylactic examinations of women at Prof. Petrov Research Institute of Oncology of the USSR Ministry of Health for the period of 1976--1980. Four annual prophylactic examinations (clinical method, thermography, mammography, biopsy) of 4823 women over 35 years of age revealed breast cancer in 51 persons (i.e. in 1.05% of the examined women). In 52.9% of cases tumors were revealed by clinical examination + mammography, in 29.4% by mammography alone, in 17.5% by clinical examination alone. In 38 out of 51 breast cancer patients (i.e. in 74.5% of cases) the detected tumors were less than 2 cm in diameter (stage I, "minimal" and preinvasive carcinomas).

14C-arginine-rich basic proteins in ascitic fluid of Ehrlich tumor.

With the use of 14C-arginine it is shown that among the ascitic fluid proteins of Ehrlich tumor there appear basic proteins rich in arginine with the content of amino acid about 14%. The amount of these proteins is about 3%. They exhibit great similarity in respect to arginine content, electrophoretic mobility to the arginine rich basic proteins isolated previously from the cytosol of Ehrlich tumor cells.

Electrophoretic analysis of radiolabeled cell surface proteins and glycoproteins of some human hemopoietic cell lines.

Cell surface proteins and glycoproteins of several human lymphoblastoid- and neoplastic hemopoietic cell lines were radiolabeled by lactoperoxidase catalysed iodination and by sodium periodate/tritiated sodium borohydride. Electrophoretic patterns of radiolabeled proteins and glycoproteins obtained by electrophoresis under denaturing conditions (SDS-PAGE) were essentially similar for all examined lymphoblastoid cell lines, with a characteristic group of major radiolabeled glycoproteins gp44 and 24,31. Characteristic, individually different and distinguishable patterns of radiolabeled proteins were observed in different neoplastic hemopoietic cell lines: T-leukemia cell line MOLT 3, erythroleukemic cell line K562, pre-B cell leukemia line NALM 6, and a promyelocytic leukemia cell line HL-60. Cell surface proteins of HL-60 promyelocytic leukemia cell line displayed some similarities to those of another myeloid leukemia cell line ML 3. Examined Burkitt lymphoma cell lines were similar to lymphoblastoid cell lines with some minor differences. Glycoprotein gp44, markedly labeled on lymphoblastoid cell lines, was absent on Burkitt lymphoma cell line Daudi. Electrophoretic patterns of cell surface proteins of blast cells from a few patients with leukemia examined simultaneously with the cell lines are described and discussed.

Immunoprecipitation of membrane proteins of cultured human sarcoma cells.

Human sarcoma associated antigens (HSAA) have previously been identified by indirect immune fluorescence in human sarcoma cells in culture using sera from patients bearing different types of sarcoma. To further characterize these HSAA, surface proteins of cultured cells were labeled with 125Iodine, [3H]-glucosamine and [35S]-methionine and solubilized. After immunoprecipitation labeled proteins were detected in immune complexes by SDS polyacrylamide gel electrophoresis and autoradiography, which allowed comparison with antigens described by other groups. A surface protein (Mr 96 000) was precipitated with sera from sarcoma bearing patients, and two glycoproteins (Mr 115 000 and 85 000) were preferentially precipitated with antisera from rabbits immunized with membranes from two human sarcoma cell lines. At least two of these proteins were found in each of five human sarcoma cell lines studied (U-4SS, U-3930S, U-20S, B-5GT and B-6FS). None of the proteins were precipitated with three human control sera, and only occasionally a faint band was observed in immunoprecipitates from control cells (B-25F, B-41B, B-42FC, U-2S, and U-393S with the immune sera. These proteins are probably some of the antigens responsible for the immune fluorescence observed in determination of HSAA. However, purification of the proteins and competition experiments are needed before this can be finally established.

Establishment of a human cell line (B-25F) derived from a fibroma of buccal epithelium.

A new cell line (B-25F) obtained from a benign polypoid fibrous lesion of the mucosa of oral cavity is described. The cells at first grew in suspension but after a month of cultivation they began to adhere and subsequently formed a monolayer typical for fibroblastoid cells. Population doubling time both in lower and higher passages was 60 h. Electron microscopy studies failed to detect any viral particles or mycoplasmas. A comparison of cell surface glycoproteins of fibroblasts (B-41FB), fibroma cells (B-25F), and fibrosarcoma cells (B-6FS) was made. These lines share common traits in their surface membranes although distinct differences among the individual lines could be detected. Karyological analysis showed that 64% of cells contained 46 chromosomes. This number veiled both diploid and pseudodiploid karyotypes. An aberrant chromosome, t(13;18?) was found. For isoenzyme comparison of B-41FB, B-25F, B-6FS, and HeLa cell lines the mobility of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PD) was employed. All these lines had LDH patterns of human cells, and a G-6-PD pattern of phenotype B except that of HeLa cells that had phenotype A.

Study of cell-mediated immunity in B77 tumor bearing rats by 51chromium release assay.

Cell immunity was followed in Lewis rats with transplanted syngeneic B77 tumors, by means of the cytotoxic 51chromium release test. Lymphocytes from peripheral blood, or from regional lymph nodes of tumor bearing animals, cocultured for 24 h with target B77 cells had an enhanced cytotoxic effect on target cells in comparison with lymphocytes of control donors. This effect of the lymphocytes still increased during 48 h of coculture.