The occurence of collagen type II in bronchogenic carcinoma.

It has been evidenced in a single case of bronchogenic carcinoma, that the proportion of collagen type II is increased in the diseased tissue. During the experimental work the collagenous stroma was isolated, subjected to cyanogen bromide cleavage and the relative proportion of individual types of collagen was judged according to the occurence of alpha1(I)CB3 and alpha1 (II)CB3 peptides. The nature of these peptides was proved by the chromatographic behavior during CM-cellulose chromatography, Ultrogel AcA 54 separation and by their amino acid composition.

Hemangiopericytoma.

Description of e hemangiopericytomas noted in a 25-(female), 24-(female) and 44-(male) year old patient. Histological examination revealed, besides characteristic appearance of tumor cells, typical arrangement of reticuline. The authors discuss the histological criteria for diagnosis and differential diagnosis and judging benignity and/or malignity of hemangiopericytomas.

Short communication: a new technique in immunosuppression of Syrian hamsters xenografted with human tumors.

Application of hyperimmune rabbit antisera against mouse lymphomas (6C3HED lymphoma, NK lymphoma) or against rat leukemic cells (RBA-34-Le leukemia) caused a considerable degree of lymphocyte depletion in hamsters.

Hyperuricaemia as an index of the response to chemotherapy in solid tumors.

A transient increase in uric acid level following administration of high doses of Cyclophosphamide in the 1st course of cytostatic therapy applied for various solid tumors (mostly lung cancer) was observed in 10 patients with measurable disease simultaneously with complete or partial (above 50%) regression of the tumor. The same effect was noted in further 5 patients, who, however, had non-measurable disease. On the other hand, the clinical course of the disease and survival were similar in these 5 patients and in the former 10 patients. No corresponding increase in the uric acid level was observed in 87 patients with similar disease and treatment, but not responding to chemotherapy and with short survival. It is concluded, that hyperuricaemia may indicate responsiveness to chemotherapy in cancer patients with non-measurable disease.

Biochemistry of drugs XXIII. Substance with antineoplastic acitivity LX. Pharmacokinetics of cis-beta-4-pentoxybenzoyl-beta-bromoacrylic acid (cis: Br, H)-3H (Penberol-3H).

After oral administration of Penberol-3H to mice with mammary gland adenocarcinoma HK, within 4 days 50% of the administered radioactivity was excreted in the urine, and 36% in the faeces. In dogs the excretion rates were slower. It was found that after oral administration of Penberol-3H to rats, radioactivity was excreted in the bile. After oral administration of Penberol-3H to mice with mammary gland adenocarcinoma, high specific radioactivities (per gram of tissue) were found in the liver, kidneys, lungs, and ovaries. Besides, it was found that after oral administration of Penberol-3H to nursing female rats the radioactivity was little eliminated in the milk, and in pregnant females its penetration into the fetuses was negligible.

Effect of cyclophosphamide on the course of cutaneous wound healing.

The healing process of cutaneous wounds has been followed with the aid of mechanical tests, autoradiographic investigation of fibroblastic reaction in the wound and method of electron microscopy. Cyclophosphamide has been found not to have any substantial effect on the favorable course of healing of the surgical wound, hence, surgical intervention combined with postoperative administration of Cyclophosphamide does not jeopardize the healing process.

Comparative investigation of DNA from mouse and Ehrlich ascites tumor cells.

Mouse DNA and DNA of Ehrlich ascites tumor have been comparatively studied in the search for changes in DNA during tumor progression. No differences were found in kinetics of homologous (mouse X mouse) and heterologous (mouse X tumor) DNA reassociation; in thermal stability of homologous and heterologous duplexes of repeated, unique and satellite DNA; in percentage of hybridization with mouse liver heterogenous nuclear RNA; in thermal stability of the RNA.DNA hybrids. The negative results suggest that the considerable evolution of transplantable tumors (both in biological properties and in karyotype) has not been accompanied by alterations of the genome which could be detected by the now available methods of molecular hybridization for studying DNA divergence. In the light of the results obtained the functions ascribed to the mouse satellite DNA are discussed.

Recovery from x-ray induced damage in human cells grown in culture.

Cells from an established human cell-line, NHIK 3025, originally derived from an early stage of cancer of the cervix, were tested for recovery capacity when irradiated with X-rays under aerobic (equilibrated with air) and extremely hypoxic conditions (O2 content less than 4 ppm). The dose-response curve obtained under aerobic conditions had a D0-value of 130 rads and an extrapolation number of 3.8. The corresponding curve obtained with a first dose exceeding that of the shoulder region of the survival curve followed 3 hours later by graded second doses, had a D0-value of 130 rads and extrapolation number 2.2. The curve obtained by measuring the survival after two equal doses of 330 rads, each with variable time intervals between the doses, showed a time dependent radiosensitivity. The dose-response curve obtained by irradiating the cells under extremely hypoxic conditions was well fitted by an exponential line up to about 2500 rads followed by a downward bend. Recovery from sublethal damage was not observed in split-dose experiments where the total doses were less than 2500 rads. For a total dose of 4200 rads, however, a split-dose effect was observed (SDR = 1.6). This split-dose effect is probably not due to Elkind repair, but is rather a consequence of the technique used.

Cellular evaluation of hypersensitivity to 2,4-dinitrochlorobenzene.

A suitable modification of the "skin window" method makes it possible to evaluate hypersensitivity reaction to 2,4-dinitrochlorobenzene at cellular level. Probands are sensitized by epicutaneous application of 0.2 ml of 1% solution of the tested substance, and after 14 days 1 drop of this solution is applied in the same manner on a freshly scraped area of the "skin window". An imprint preparation is taken 48 hours later and examined microscopically. Presence of lymphoid elements, eosinophils and basophils points to a normally developed hypersensitivity reaction. Presence at the same time of rather great amounts of large monocytogenic macrophages and neutrophilic granulocytes is an expression of an unspecific inflammatory reaction taking a normal course. Suitability of the described procedure for routine application is discussed.

Chromosomal characteristics and non-random distribution of sister chromatid exchanges in lymphoblastoid cell lines isolated from acute leukemias.

Serial cytogenetic characteristics of four lymphoblastoid cell lines derived from the peripheral blood of four patients with acute leukemia are presented, including G and C banding techniques and the analysis of sister chromatid exchanges by means of FPG technique. One of these lines turned into predominantly diploid although originally pseudodiploid, while the other three changed from diploid to heteroploid ones during prolonged cultivation, with the appearance of a marker chromosome in one of them. The mean number of sister chromatid exchanges per mitosis (9.6) did not differ from the control value obtained in short-term cultivations of lymphocytes of healthy subjects. All lines showed a significantly increased number of sister chromatid exchanges in the B group chromosomes. The contribution of FPG technique for the study of cell kinetics is briefly discussed.

Cytologic diagnosis of malignant melanoma.

Cytology in malignant melanoma of the skin is a useful method, which alone yields in many cases enough diagnostic informations for a therapeutical approach. It is of a special importance in cases with marked anisocytosis of cells and with large, sometimes polynuclear cells and with cells with melanotic pigment. In cases with middle-sized apigmented cells of epitheloid appearance it should be always completed by biopsy, which, in these cases predominates over cytology. Combination of both methods brings a more substantial elucidation of diagnosis.

Estimation of StilbostatR treatment efficiency in breast cancer by thermography.

Administration of Stilbostat to postmenopasual women with advanced breast cancer markedly improved the clinical state in about 65% of the cases and concomitantly decreased the cutaneous thermic values at the level of the lesions showing a tendency to equalize the values recorded for the symmetrical healthy cutaneous zones. The thermographic test is proposed as a means of estimating the Stilbostat treatment efficiency in breast cancer.

Isolation and properties of gamma-glutamyltranspeptidase from Morris hepatoma 5123D.

gamma-Glutamyltranspeptidase was purified 600-fold from Morris hepatoma 5123D by six-step procedure. Its apparent molecular weight estimated by centrifugation in sucrose gradient with Triton X-100 amounts to 108 000. Some dipeptides particularly glycylglycine and several amino acids considerably increase the enzyme activity but L-serine with borate decreases it. Usually transfer activity of the enzyme towards gamma-L-glutamyl substrates was much higher than hydrolytic. The best substrate for the hepatoma enzyme is reduced glutathione.

Experiments to increase the selectivity of tumor chemotherapy by means of in vivo activation of transport forms of cancerostatics by exogenous enzymes.

A significant tumor damaging effect (growth inhibition) on transplanted syngeneic sarcoma in mouse was obtained by means of pH-dependent activation of a transport form of a cancerostatic drug by an enzyme foreign to the organism. This effect was achieved by combined administration of 8-0-(alpha-L-arabinofuranosyl)beta-peltatin-A as a transport form of beta-peltatin-A and the exogenous enzyme alpha-L-arabinofuranosidase from Aspergillus niger and additional increase of the acidity of the tumor by injection of glucose. The combined application of the transport form plus enzyme showed a more favorable effect on selectivity than free peltatin when a quantitative comparison was made between the tumor growth inhibition and the damage to the blood picture.

The effect of growth-promoting alpha-globulin (GPAG) on the incorporation of exogenous DNA into L-cells. I. Incorporation of isologous and heterologous 3H-DNA.

Isologous and heterologous 3H-DNA (optimum concentration 25 microgram/ml of medium) are incorporated into L-cells from the medium during short-term incubation (up to 60 min). The incorporation of DNA is stimulated by a protein complex from calf serum--the growth-promoting alpha-globulin (GPAG) in the concentration 0.8 mg/ml of medium), which is rapidly taken into cells by pinocytosis. GPAG increases the frequency of incorporation of exogenous 3H-DNA into L-cells and the quantity of DNA incorporated. The quantity of 3H-DNA incorporated is further increased on the incubation of L-cells in a medium containing the complex 3H-DNA + GPAG, which is formed by the joint preincubation of the two components at 37 degrees C (20 hr); under these conditions the quantity of DNA incorporated is 150% greater than when 3H-DNA is used alone. GPAG acts as an activator of exogenous DNA transfer, stimulates its pinocytosis by the cells, and at the same time accelerates its intracellular transfer to the cell nuclei.

Localization of alpha-fetoprotein by immunofluorescent method during induction of rat liver tumors by 3'-methyl-4-dimethylaminoazobenzene.

Localization of alpha-fetoprotein (alpha-FP) has been followed in hepatal tissue and tumors during induction of primary hepatomas with the aid of 0.12% 3'-Me-DAB (3'-methyl-4-dimethylammoazobenzene) in Wistar rats. The indirect immunofluorescence method was used for the localization of alpha-FP positive cells. During the course of carcinogenesis, alpha-FP in serum was detected by means of the crossing over immunoelectrophoresis. This study has yielded the following results: Alpha FP positive cells resembling small hepatocytes occurred dispersed and in groups beginning with the 5th week of a carcinogenic diet until the appearance of tumors. No alpha-FP positive oval cells have been found. Alpha-FP positive cells were always found in rats with alpha-FP positive serum, but they were rarely present in rats with alpha-FP negative serum. From the 10th week, tumors of the cholangiohepatoma type began to be formed in which variously scattered alpha-FP positive cells of the type of small hepatocytes were present, with the serum being negative. Between week 14 and 21 hepatoma nodules began to be formed. At week 21 frequent alpha-FP positive cells close to normal hepatocytes were observed both singly and in groups. These are considered to be the sites of developing tumor nodules. In all the hepatoma nodules, the number of positive tumorous cells and the intensity of fluorescence proved to be directly proportional to alpha-FP concentration in serum.

Immune response to Rauscher virus-induced leukemia in DBA mice. II. Correlation between antigenic expression and pathogenesis.

Previous studies have demonstrated that the Rauscher virus induces a biphasic erythroleukemia in DBA mice, and the regression of the disease is connected with the appearance of antibody-dependent cellular cytotoxicity. In the present work, attempts were made to reveal the mechanism leading to the lethal exacerbation of the leukemia. In the sera of leukemic mice soluble tumor-specific antigen could be demonstrated in the stages of early progression and regression but not in the stage of exacerbation. The antigen was present in form of immune complexes with free antibodies in excess. The emergence of a new population of leukemia cells has been observed during the stage of regression. On the surface of these cells the antigen receptor sites were masked by sialic acid which resulted in the loss of immunosensitivity and immunogenicity.

Complement-fixing soluble antigen from rat lymphoma (C58NT)D. Preliminary characterization of the antigen and some positive human sera.

A soluble complement-fixing antigen prepared from the Gross virus-induced rat lymphoma (C58NT)D reacted in complement-fixation with the sera of some patients with different malignancies (particularly acute leukemia) and with the sera of some healthy individuals. Results of gel filtration of crude (C58NT)D antigen on Sephadex G 200 indicate that the antigenic activity is eluted with the void volume of the column, i. e. in fractions corresponding to proteins of low molecular weight. Gel filtration of some positive patient sera indicate that complement-fixing activity of tested sera is associated with fractions corresponding to IgM and/or IgG.

Lower gastric secretion and higher incorporation of orotic acid into liver RNA in rats treated with 5-azacytidine and cycloheximide.

Out of different azapyrimidines tested for their ability to affect metabolism of orotic acid in the liver of rats kept on food only 5-azacytidine resulted in the enhanced incorporation of orotate into liver RNA following 24 hr pretreatment. Similar effect was observed also in cycloheximide-treated animals. No stimulation of orotic acid utilization following 5-azacytidine or cycloheximidine treatment was observed in the liver of starved animals. Both drugs (but not other pyrimidine analogues tested) depressed markedly gastric secretion in rats and caused decreased evacuation of the stomach. The decreased secretion of pepsin and lower gastric acidity resulting in drug-simulated starvation of the treated animals are discussed in relation to the enhanced uptake of orotic acid into liver RNA.

Effects of 5-aza-2'-deoxycytidine on DNA synthesis in mouse lymphatic tissues.

5-Aza-2'-deoxycytidine-3H administered i. p. to mice is preferentially incorporated into nucleic acids in the spleen and thymus. The labeling of the spleen is maximal during first hours after drug administration and decreases rapidly thereafter. Following administration, 5-aza-2'-deoxycytidine is metabolized giving rise to several new compounds. The incorporation of 5-aza-2'-deoxycytidine is blocked by the simultaneously administered deoxycytidine to a lesser degree than that of deoxycytidine by the analogue. Maximal inhibition of deoxycytidine incorporation by the drug was observed in the spleen; the incorporation of thymidine under similar conditions was diminished only about 20%. However, pretreatment of the animals with the analogue resulted in a strong inhibition of thymidine incorporation (about 80%) both in the spleen and the thymus. Simultaneously the activity of thymidine and thymidylate kinases in cell-free spleen extracts was markedly depressed.

The retrovirus particles in human myeloma cells RPMI8226: morphological, biochemical, immunological and infective transmission studies.

The retrovirus designated RPMI8226V (isolated from human myeloma cells RPMI8226) has been characterized with respect to its morphological, biochemical and immunological properties as well as its propagation in various animal and human cells. The myeloma cells RPMI8226 produce intracytoplasmatic A-type particles and extracellular particles. The extracellular particles have been classified as immature particles with translucent core center, typical mammalian C-type virus particles and C-type particles with intermediate membrane. However, the budded particles in secondarily infected human neoplastic cells contained complete doughnut-shaped nucleoids. This type of budding is rather characteristic for B-type particles. The 3H-uridine labeled RPMI8226 viral particles have a buoyant density 1.17 g/ml in sucrose gradient containing high molecular weight RNA and the distribution of viral structural proteins in SDS-PAGE is characteristic for oncornaviruses. The internal structural proteins according to MW are ranged from 13 000 to 30 000 daltons. The virus contains a magnesium-dependent reverse transcriptase. The cellular homogenate and viral concentrate from RPMI8226 cultures do not react with antibodies against ALSV, MuLV, FeLV, RD114, MP-MV and SiSLV. The only reaction was scored with anti BLV antibodies. However, anti BLV serum inhibiting the reverse transcriptase activity of BLV to 60% does not cross-react with the reverse transcriptase of RPMI8226V. In contrast to BLV concentrates, neither XC nor KC cells show syncytia formation by RPMI8226V. The RPMI8226V replication is restricted to human tumor and normal human glia-like cells. The possible origin of the virus is discussed.

Checking of carcinoma patients with the leukocyte migration technique (LMT) under agarose.

Leukocyte migration tests under agarose (Clausen technique) were performed in 28 patients tentatively diagnosed as having any malignancy with the use of a 3 M KCl-extract panel prepared from bronchogenic, gastric, colonic, renal, and mammary carcinoma, corresponding normal tissues, carcinoembryonic antigen (CEA), and human encephalitogenic protein (HEP). 17 out of 22 proven carcinoma patients showed sensitization by reaction with optimal concentrated KCl-extract of cancer from the same organ type as their own tumor. In some cases positive reactions could be observed also with normal tissue antigen (NTA) of tumor organ type (7/22) or with an additional carcinoma extract of organ type differing from patients own primary tumor (8/22). Gastrointestinal carcinomas, especially, showed sensitization to CEA (7/12) contrary to nongastrointestinal carcinomas (1/10). With HEP no positive reactivity could be found (0/10). With the use of tumor antigen panel (5 antigens) only few positive reactions (MI less than 0.80 or greater than 1.20) could be observed in 6 patients with nonmalignant diseases (1/30 tests) and 8 healthy blood donors (1/40 tests). A widespread individual screening program using tissue antigens for patients suspected of malignancies could give a pattern of reactivities and improve the recognition of cell-mediated sensitization against tumor tissues.

Human urinary bladder carcinoma cell line (T24): immunological studies and search for oncornavirus in T24 cell population and derived clones.

Cytotoxicity of mononuclear cell (MNC) preparations isolated from peripheral blood of patients with urinary bladder carcinomas of a transitional (BTCC) or squamous (BSCC) type and from blood of control subjects was examined on the target cells of T24 cell line. In pilot experiments the percentage of cytotoxic MNC preparations was found to be similar in patients with tumors of BTCC (European, 56%;African, 60%) and BSCC (54%) type. Cytotoxicity of MNC prepared from control subjects was detected in 10% of cases or less. These data suggest an antigenic cross-reactivity between urinary bladder carcinomas of transitional and squamous cell type. The cytotoxic MNC from peripheral blood of patients with BTCC and control subjects were fractionated by adherence in nylon wool columns, and the cytotoxicity of adherent and nonadherent MNC subpopulations was examined. In the majority of cytotoxic MNC preparations from both donors with and without BTCC, the cytotoxic activity was associated with nonadherent MNC. Comparison of immunosensitivity of T24 cell population with the cloned T24 subpopulations indicated that clones with various digrees of immunosensitivity are present in the T24 cell population. However, immunosensitivity of the clones was never higher than the immunosensitivity of the T24 cell population. A search for oncornavirus particles produced spontaneously or after treatment with virus production activators performed with regard to the possible association of oncornaviruses and antigens responsible for cell-mediated cytotoxicity gave thus far negative results.

Characterization of murine hepatoma BW7756. II. Comparison of adult, fetal, and neoplastic liver soluble antigens.

Isolation of alpha-1 fetoprotein.

A suitable method of the isolation of alpha-1 fetoprotein for the needs of enzyme immunoassay of this oncofetal antigen is described. By combining isoelectric focusing and "indirect" affinity chromatography the preparation of alpha-1 fetoprotein was obtained that was not contaminated with IgG, contrary to the isolation performed by means of "direct" affinity chromatography on a carrier with coupled anti-alpha-1 fetoprotein antibodies, or other immunochemical methods that usually yielded contaminated preparations. Neither disc electrophoresis in PAA gel, immunoelectrophoresis, double radial immunodiffusion, nor biological experiments revealed any traces of ballast proteins in the resulting preparation; it seems suitable both for the preparation of monovalent antisera of a sufficient avidity, and as a standard for enzyme immunoassay.

Detection by indirect immunofluorescence (IF) of human sarcoma associated antigens in established sarcoma cell lines.

Mitochondrial ATPase of Zajdela hepatoma. V. Mitochondria of Zajdela hepatoma contain membrane sectors of ATPase complex unassociated with F1.

Relative content of membrane sectors of ATPase complex in rat liver and Zajdela hepatoma mitochondria and the ability of mitochondrial membrane of the two sources to bind isolated soluble (F1) ATPase were examined. Approximately equal concentrations of oligomycin were required for 50% inhibition of ATPase activity in submitochondrial particles of rat liver and Zajdela hepatoma indicating practically identical content of membrane sectors of ATPase complex in both types of mitochondria. As detected by the increase in oligomycin-sensitive ATPase activity of submitochondrial particles incubated with isolated F1, the submitochondrial particles of Zajdela hepatoma in contrast to those of rat liver were able to bind specifically considerable amounts of exogenously added F1. The results indicate that mitochondria of Zajdela hepatoma contain membrane sectors of ATPase complex unassociated with F1 but capable of association with this enzyme.

Sensitivity of TSTA and species-specific cell membrane antigens of tumor cells to glutaraldehyde treatment.

In vivo immunogenicity and in vitro species-specific membrane antigens in tumor cells treated or untreated with glutaraldehyde (GA) were studied. Two different syngeneic Syrian hamster transplantable tumor cell lines (spontaneous liver cancer and SV40-induced sarcoma) not only lost immunogenicity after GA treatment but were responsible for enhancement of test-tumor growth in immunized animals. In vitro mixed hemadsorption test used for determination of species-specific membrane antigens in Syrian hamster, green monkey and interspecies hybrid cells revealed drastic alteration of antigens on the membrane of cells treated with GA.

Immunochemical and clinical characteristic of series of 516 paraproteinemic patients.

A series of 516 cases of paraproteinemia was analysed. According to the class of paraprotein, there were 336 cases of paraprotein IgG (65.1%), 101 paraprotein IgA (19.6%), 53 paraprotein IgM (10.3%) and 1 case of paraprotein IgD (0.2%); only light chains were found in 15 cases (2.9%) and doubled paraproteins in 10 cases (1.9%). The average age of the series was 64.2 years. Clinically, there were 315 cases of myeloma, 24 cases of Waldenström's macroglobulinemia, 126 cases of facultative paraproteinemia accompanying another disease, 16 cases of paraproteinemia in healthy persons and in 10 cases of paraproteinemia no precise diagnosis was made.

Simultaneous occurrence of IgM-lambda paraprotein with pyroprecipitation properties and cryoglobulin in the serum of a patient with Waldenström's macroglobulinemia.