Effect of hydroxyurea and vinblastine on the proliferation of the pluripotential stem cells.

The population of the pluripotential hemopoietic stem cells in mice, i. e. those cells forming colonies in the spleens of lethally irradiated mice (colony forming cells CFc) is proliferating relatively slowly. After a partial damage the population regenerates which is achieved by means of the increased proliferation rate. The effect of damage caused by different doses of hydroxyurea or vinblastine on the proliferation of the CFc has been investigated. CFc population was measured in femur bone marrow after grafting the sample of the bone marrow into the lethally irradiated mice recipients (spleen colony method). The proliferation rate was estimated either according to the magnitude of the fraction of cells synthesizing DNA in the S phase of the cell cycle, or according to the sensitivity of the population to the repeated injections of vinblastine. Data showed that even after very minute damage caused by hydroxyurea the stem cells started to proliferate intensively. The effect was dose dependent. The comparable damage caused by vinblastine had a significantly weaker effect on the proliferation of the stem cells. From the results it is concluded that the proliferation response of the pluripotential stem cells depends on two factors: one being the extent of the damage caused to the hemopoietic tissue and the second the position of the killed cells in the cell cycle.

Cinemicrographic analysis of the cellular changes of the stabilized cell line HeLa provoked by l-aminoadamantane.

The effect of antiviral compound l-aminoadamantane on HeLa cell culture has been studied. Morphological changes were observed at the concentration 300 and 500 mug/ml. The concentration of 150 mug/ml exerted a delay in the process of cell adhesion and dilatation (spreading).

Human malignant melanoma cells: morphological and immunological variations.

When cultured in vitro, human malignant melanoma cells proliferated in two patterns: In one the cells were polygoneal or spheroidal unequal in form and size, whereas in the other, the cells were elongated, fusiform, poorly delimited, with various degrees of atypism. Antisera to each of these two types of malignant melanoma cell cultures were raised in rabbits. The antiserum cytotoxic activity was assessed against 10 cultures of malignant melanoma cells, 10 mammary carcinoma, 2 fibrosarcoma and 10 normal skin fibroblasts. Prior and at intervals post surgery, the cytotoxic activity of serum from 10 patients with malignant melanoma was assessed against the above cell cultures. Based on their cytotoxic activities, the sera drawn from patients with melanoma thirty days after surgery had the highest cytotoxicity and could be classified into two groups similar to the rabbit antisera. Glycoprotein fractions were isolated from 3 M KCl extracts of the two melanoma cell types, i.e. from patients ShA and ZBJ. These glycoproteins stimulated 14C-2-thymidine uptake and DNA-polymerase activity of peripheral blood lymphocytes from post surgery melanoma patient. The glycoproteins had no effect on peripheral blood lymphocytes from normal donors.

Immunosuppression and Carcinogenesis: effects of azathioprine on induction of sarcomas by 3-methylcholanthrene.

The effects of Azathioprine (AZA) on the carcinogenesis with 3-Methylcholanthrene (MC) are described. When AZA is given before MC, there is a shortened tumor latency period. When AZA treatment followed MC injection a lengthened latency period was observed. When AZA treatment is given both before and after MC injection, intermediate results were observed. It is concluded that the action of AZA treatment on tumor development depends on the time relationship between this treatment and the injection of the carcinogen. In particular, it is suggested that to obtain an enhancing effect on tumor development, probably by immunosuppression, AZA must be effective at the time or shortly after administration of the carcinogen. When AZA is administered later, an inhibition of tumor development, probably a cytostatic effect becomes prominent.

Dextramycine (the dextraisomer of chloramphenicol) as an inhibitor of the induction of lung adenomas in mice.

Dextramycine as well as leftrotating isomer (chloramphenicol--levomycetin) diminished the blastomogenic effect of urethane which is known to induce lung adenomas in mice.

Influence of stress and of endocrine imbalance on the experimental metastasis.

The influence of some stress agents applied during variable periods of time and under various modifications of the endocrine status was studied on the lymphotropic metastases of the 256 Walker carcinosarcoma, grafted intratesticulary in rats. Different reactivity of metastases, when compared to that of primary tumors was found in relation to the action of some compounds influencing the hormonal balance. Stress enhances the primary tumor development, while the metastatic growth is inhibited. However later on, sudden enhancement of the metastasis growth was observed. Adrenalectomy, as well as testosterone, thyroxine, of Stilbostat administration inhibit the metastasis, whereas cortisone, or antithyroid drug administration enhance the metastasis. The in vitro study of the tumor cell proliferation suggests some immunemorphological involvements in the development of this process.

Hamster apigmented melanoma as a model for screening of potential antineoplastic drugs.

The authors treated the apigmented melanoma IC-Sofia in hamster with various doses of the following cytostatics: Cyclophosphamide, 6-Mercaptopurine, Vinblastine and Actinomycin D for 7 consecutive days. Determination of the tumor growth inhibition on the 8th day (early test) on the basis of tumor weight in the animals treated and untreated with cytostatics indicates that the tumor used is more sensitive to the effect of Cyclophosphamide and 6-MP and less to Vinblastine and Actinomycin D. Nevertheless sensitivity to cytostatic action is high enough for each of the cytostatic agents, depending upon the applied dose. This gives grounds to the authors to assume that hamster apigmented melanoma IC-Sofia is an adequate model for testing and evaluation of antitumor drugs.

Serum immunoglobulin levels in cancer patients. III. Immunoglobulin levels and metastases of malignant tumors.

The levels of IgG, IgA and IgM were followed in 93 patients with metastases of various tumors in the liver, bones, lymph nodes, lungs and with the generalization of tumor in multiple organs. A significant increase of IgG was noted in those patients where liver, bones and lymph nodes were involved, compared to other tumor groups as well as with the values considered as normal range. A significant rise of the IgA class was observed in patients with liver metastases compared to cases with the generalization of tumor. In all groups the mean levels were scattered above the upper limit of normal values. The mean levels of IgM class did not exceed the upper normal limit, but in the group of bone metastases a significant rise was noted compared with the group of generalized tumors.

Serum immunoglobulin levels in cancer patients IV. The influence of therapy on the levels of IgG, IgA and IgM.

The levels of immunoglobulins (IgG, IgA, IgM) were measured in the blood serum of 232 patients with malignant diseases subjected to therapy with irradiation, cytostatic drugs, hormones and active immunotherapy. The values of two samples were compared before and after therapy. The local irradiation with usual doses and technique did not significantly influence the Ig levels. On the contrary, the levels of all three Ig classes decreased after chemotherapy, but only the IgG decrease was significant. A rise of Ig followed active immunotherapy and hormonal therapy but these changes were not significant.

Endocrine-metabolic criteria for distinguishing two pathogenetic types of breast cancer.

Hypothalamic threshold of sensitivity to homeostatic inhibition was studied by means of the dexamethasone test of inhibiting excretion of steroid hormones in 47 patients with the primary breast cancer of different age groups. Before the dexamethasone test and after it, blood specimens were taken for the following tests: daily variation of blood sugar or sensitivity to insulin action, cholesterol, lipoproteins, phospholipids, uric acid, growth hormone and insuline levels, and level of corticosteroids. On the base of the results obtained all the patients could be divided into two groups: 1. patients with signs of elevated threshold of hypothalamic sensitivity to inhibition and 2. patients without such signs. The breast cancer developing against the background of endocrine metabolic disturbances characteristic of advanced age is on the whole prognostically more favorable than tumors in women who have no disturbances of that kind. However, metabolic shifts characteristic of ageing obviously play an important role in creating a background that promotes higher incidence of mammary cancer in elderly subjects.

Chemotherapy and "second-look" procedure in the treatment of ovarian carcinoma.

A combination of chemotherapy with a "second-look" operation has its place in a long-tern treatment of ovarian cancer of stage III, though it will hardly improve the long-term results--the 5-year survival in this stage. Ocassionally, this combination may prove useful and even decisive for prolonging life and even bringing about recovery. This would apply to less advanced, better cases of stage III and to carcinomas with a lesser biological malignancy. "Second-look" operations may be indicated not only in an improvement of the local finding as has been reported up to now by several authors, but also if this local finding has not improved but the patients overall condition is satisfactory or has not deteriorated since the first laparotomy, especially if the latter had been performed at another hospital. The time for the second-look operation must be determined individually according to the clinical course.

Results of radiation therapy and a combination of radiation and surgery in the laryngeal cancer.

A total of 348 out of 349 patients with laryngeal cancer were treated over the period 1960-1970. Radiation therapy alone was used in 262 of them and surgical intervention with postoperative irradiation in 86. Results in the form of a five-year survival rate in the group treated solely by irradiation were superior by 12% in the case of glottic than in that of supraglottic carcinoma (48.8% and 36.8% respectively). In the group of patients operated upon and subsequently irradiated, relatively satisfactory results were obtained in the advanced, i. e. the III stage of supraglottic carcinoma (5-year survival in 61.4%). The overall 5-year survival rate in the patients operated upon and then irradiated proved to be better by 24.5% in supraglottic and by 4.1% in the glottic carcinoma, than in patients subjected solely to irradiation. Of 27 cases of relapses, 6 patients were successfully treated by laryngectomy. Post-irradiation complications involved most frequently minor cases of endolaryngeal edema; among the more serious ones were 2 cases of radiation myelitis. The discussion on the problem of radiation dosage in cancer of the larynx, the treatment of regional metastases and combined surgico-radiation therapy.

Different sensitivity to antibiotics of the established intermitent in vivo--in vitro tumor cell lines in comparison with the primary cultures.

From original strains of tumors growing only in vivo (I) new lines of strains (II) were obtained, the cells of which proliferated readily both in vitro and in vivo. Sensitivity in all the new strains (II) generally increased but in varying degrees as against individual cancerostatic drugs, as evident from the given ID50 values and sensitivity indices. In addition to an enhanced sensitivity of the strains to inhibitors, their ability to utilize precursors of the synthesis of nucleic acids and proteins in a linear dependence during the course of 24 hours has also been determined.

Biopharmacological investigation of 6,6'methylene-bis/2,2,4-trimethyl-1,2-dihydroquinoline (MTDQ) a radical binding antioxidant of secondary amine type.

The evidence of free radicals in tumor tissue and the possibility of their experimental influencing by means of antioxidants justifies the search for new pharmacological groups of tumor inhibiting agents. The authors synthesized a sterically inhibited, heterocyclic, bifunctional, non-toxic radical binding antioxidant of secondary amine type. The structural change is interpreted with the possibility of recombination characteristic for secondary amine groups and the double chain closing effect. The conjugation effect secures mobility of the hydrogen atom. The compound contains 10 to 12 per cent dimer-trimer of higher biological activity than monomers, due to higher molecular weight. An additional pharmacodynamic advantage compared to hitherto known antioxidants consists in its lower vapour volutility and the biradicality.

Effect of "3-oxauracil" (2,3-dihydro-1,3-6H-oxazine-dione) in L1210 leukemia.

2.3-Dihydro-1,3-6H-oxazine-dione ("3-oxauracil") was tested against L1210 leukemia. The compound exhibited significant growth-inhibiting effect against this tumor both in vitro and also in vivo following daily intraperitoneal application.

Chromosomal changes in rat pituitary and bone marrow induced by long-term estogen administration.

Pituitary weight, mitotic index and chromosomes were studied in male rats following a single or repeated dose of estradiol-benzoate for a total period of 210 days. Estrogen-induced pituitary hyperplasy eventually resulted in large pituitary tumors in some animals. The mitotic index increased until day 90, when it diminished, with chromosomes partly replaced by chromatin clusters. Most of the cells of the hyperplastic pituitaries maintained a normal diploid karyotype. However, a limited number of abnormal stem-lines with marker chromosomes appeared at a relatively early stage of the hyperplastic reaction of the pituitary. Later the tumors were characterized by an increase of number of aneuploid cells. In the bone marrow estrogens significantly lowered the mitotic index without any detectable change in chromosomal morphology.

Host-tumor relationship. XXXIV. Hyaluronidase activity and hyaluronidase inhibitor in the serum of patients with malignant tumors.

Current determinations of the hyaluronidase activity and hyaluronidase inhibitor in the serum of patients with a tumor disease failed to show a correlation between a lowered hyaluronidase activity and increased inhibitor level. The serum fraction that contained the inhibitor and which had been obtained by gel filtration of Sephadex G-200 had no in vitro inhibitory effect on the serum fraction containing hyaluronidase. Hence, the decreased serum hyaluronidase activity in these patients is not due to an increased levles of hyaluronidase inhibitor.

Some data concerning immune processes in concomitant tumor immunity experimental models. Comparative in vivo and in vitro investigation I. In vivo experiments.

Concomitant tumor immunity evinced by C57BL/6 mice, bearing a MC-induced sarcoma, was evaluated by graded challenge doses for different primary tumor sizes (2-3,4-6,8-12% tumor weight of the total body weight TW/TBW). 100% of mice bearing tumors, representing 2--6% of total body weight, rejected doses from 0.2--1 X 10(4) cells. The gradual curtailment of the concomitant tumor immunity, depending on increasing TW/TBW ratio, could be evaluated, using adequately increasing challenge doses. The immune equipotency of the whole s.c. body area, the failure to modify the concomitant tumor immunity by drainin node excision and the demonstration of its dependency upon the total challenge-dose and its independency upon fractionated multilocular inoculation of the challenge, showed clearly that the concomitant tumor immunity is a local expression of general immunity. The experimental model allows a valuable biological assessment of the tumor-beareer immune status and represents likewise an adequate tool for immunotherapeutic effects estimation.

Some data concerning immune processes in concomitant tumor immunity experimental models. Comparative in vivo and in vitro investigations II. In vitro experiments.

The humoral and cellular immune status of C57BL/6 male mice and "R" male rats bearing MC-induced sarcomas were investigated in vitro, using 51Cr-releasing and mixed hemadsorption assays. Analysis was performed on mice subjected to concomitant tumor immunity (CTI) model experiments or bearing primary tumors of different sizes, that was done also in rats. Chromatographic fractions, assumed to contain tumor specific antigens, antibodies or antigen-antibody complexes were identified by their absorption capacity upon specially prepared syngeneic immune sera. Free antibody and antigen-antibody complexes, accompanied with a weak cell mediated immunity (CMI) were correlated with efficient CTI in 2--3% TW/TBW tumor bearers. Homoral immunity (HI) showed sometimes an important increase after challenge administration. High levels of free antigen and antigen-antibody complexes, lack of antibodies and CMI were correlated with CTI absence in huge tumor bearers, representing 30--37% of total body weight. Conditions determining variability of results reported by different authors and the possible mechanisms by which serum immune factor may impair the tumor bearer's immune status are discussed.

Human urinary bladder carcinoma cell line (T24) in long-term culture: chromosomal studies on a wild population and derived sublines.

Karyotype changes of human urinary bladder carcinoma T24 cell line were studied in the course of 5-year in vitro cultivation. It has been found that the modal number of chromosomes gradually shifted from hypotetraploidy to triploidy during long-term cultivation. The number of endoreduplications was decreasing simultaneously and minute chromosomes occurring in a number of 4--5 in early passages appeared only rarely in later passages. Using conventional Giemsa staining, persistent marker chromosomes were detected in the wild population as well as in the eight sublines derived from T24 cells by cloning. The marker chromosomes were: metacentrics, subtelocentrics, telocentrics and long acrocentrics. Occasionally, there were found dicentrics, double-minutes, breaks and pulverization. The most characteristic marker was the metacentric chromosome the length of which corresponded approximately to the arm length of the chromosome No. 1. The metacentric chromosome in a number of 1--3 was present in 100% of T24 cells of the wild population and derived sublines at all passage levels examined.

Cell proliferation kinetics in gynecologic cancer.

The toxicity and antitumor activity of three individual fractions of lectins from Ricinus communis seeds.

Lectins from Ricinus communis seeds (RCL) have been resolved into three electrophoretically distinct fractions (alpha-RCL, beta-RCL, psi-RCL) by ion exchange chromatography on Watman CM-32 cellulose. The toxicity to mice and antitumor activity against murine lymphoma NK/Ly of pure fractions were compared. Optimal effect was achieved with alpha-RCL, Which possessed the lowest toxicity (1000 micron g/kg) and significant antitumor activity (63% inhibition of tumor growth).

A study of plasma CEA by a direct radioimmunoassay method in 1704 cases.

The authors have used an original method of direct radioimmunoassay of plasma CEA for testing plasma of 1704 patients affetected by various neoplastic and non-neoplastic diseases. The percentage of positive results in blood from patients with adenocarcinoma of the gastroenteric tract was 66.8%. The positivity in other non-neoplastic diseases was 2.7%, except for liver cirrhosis and other chronic hepatopathies, which showed 29.6% of positive reactions. The test should be clinically useful in the differential diagnosis of gastro-enteric carcinoma.

A new substance effective against transplantable tumors in vivo: L-cystine-bis-(N,N-beta-chloroethyl)-hydrazide.

It is shown that L-cystine-bis-(N,N-beta-chloroethyl)-hydrazide-hydro-bromide possesses strong (50-100%) inhibitory effect in vivo against myeloma P-8, carcinosarcoma Walker, lymphosarcoma Pliss, sarcoma Yoshida, sarcoma Jensen and sarcoma 180 in doses 5-12 mg/kg/day. No suppression of the growth of Ehrlich ascites tumor was observed. The acute toxicity (LD50) of this substance on mice and rats is 71 mg/kg and 47 mg/kg respectively.

Cardio-toxicity of Daunomycin and Adriamycin.

Our aim was to check the possibility of modifying the cardio-toxicity of Daunomycin and Adriamycin on guinea pig atria in vitro, through a reduction of calcium concentration in the medium. In a first series of tests, left and right atria still beating were put in a bath containing Tyrode solution. In order to test the influence of hypocalcic conditions on the negative inotropic effects of the two drugs. Tyrode solution contained either the usual amount of calcium or only 50% of it. In a second series of experiments isolated tissues were electrically stimulated in Krebs-bicarbonate solution. In order to avoid the spontaneous activity of the preparations, only isolated left atria were used. Again the medium contained either the normal quantity of calcium or only 50%. The reduction of calcium concentration in the medium results in a statistically significant potentiation of the negative inotropic activity of the two drugs.

VM 26 (4-demethyl-epipodophyllotoxin-beta-d-thenylidine glucoside) in the treatment of urinary bladder tumors.

VM 26, a semisynthetic podophyllin derivative (4-demethyl-epipodo-phyllotoxin-beta-d-thenylidine glucoside) has been tested in 67 patients with urinary bladder tumors. In 25 patients with stage T 1 significant prolongation of the disease-free interval was reached. In 25 cases with state T 3-4, the effect of VM 26 was not favorable.

Combination of regional chemotherapy and mastectomy in the treatment of carcinoma of the breast. Five-year results.

Thirty-six women with cancer of the breast were treated with infusion of cytotoxic drugs into the first portion of the subclavian artery adjuvant to radical or simple mastectomy. The patients were followed up for five years and the results were compared with a matched group of patients treated by mastectomy alone. There was no difference between the two groups in the overall mortality at the end of the fith year. The survival rates of patients with stage 3 carcinoma receiving adjuvant chemotherapy were in the first three years significantly better than those of the controls. No impairment of the wound healing was observed and the systemic side effects of the cytotoxic drugs were unimportant. In two patients serious neurologic complications developed and one patient died of pulmonary abscess.

Spermatic vein phlebography in patients with testicular tumors.

The performance of funicular lymphography and spermatic vein phlebography during orchiectomy in patients with testicular tumors demonstrates the close proximity of the spermatic vein to the lymph nodes draining the testicles. In the case of metastatic lymph node involvement the spermatic vein phlebography may add supplementary diagnostic information. The examination was proved successful in eight patients with malignant testicular tumors.

Some remarks to the histological classification of malignant melanoma of the skin and its prognostic values.

Histological classification of human skin malignant melanoma as recommended by WHO melanoma group renders the first useful orientation in regard to the biological behavior of the tumor, but it cannot give any further clinical outlook. It would be therefore desiderable to complete the histological examinations with enzymatic and immunologic examinations performed during the treatment of the patient and thus try to gain a basis for a prognostic evaluation.

Morphological analysis of malignant melanoma cases with prolonged survival.

Retrospective morphological analysis of 145 malignant melanoma cases was carried out to prove the prognostical importance of some morphological characteristics of the tumor. The cases were divided into two groups according to the survival time. The majority of the tumors with long survival time showed little capacity of invasion, while those with short survival time invaded at deeper levels of the dermis. Other features seemed to be of no prognostical relevance. Difficulties in determining the depth of invasion exactly were also discussed.