Neoplastic progression evidenced in the L929 cell system. II. In vitro growth properties and biochemical characteristics of cell variants with different malignant behavior.

Using a culture cell system, derived from a low-tumorigenic strain of L929 mouse fibroblasts, including cell variants with different malignant behavior in vivo, we have tried to dissociate a group of biological and biochemical transformation-associated properties with respect to the process of neoplastic progression. It is shown that morphologic changes, nonalignment in vitro, and growth in soft-agar are not sufficient alterations for in vivo malignant behavior and, also, not useful indicators for further neoplastic progression. The rate of hexose uptake should also be considered as unrelated to this process. The increase in tumorigenicity, but without the concomitant development of metastasis ability of the cells, was associated with the decrease of a 160k cell surface protein and the diminution of the tumor dose 50%. Finally, further alteration in morphology and the appearance of a 177k cell surface protein were associated with the development of metastasis ability, in an early stage; loss of fibronectin and the ability to grow as macrocolonies in increasing concentrations of deoxyglucose appeared as associated to more advanced stages of neoplastic progression. On the whole, the approach of taking the in vivo behavior, and not the in vitro properties, as end-point for the study of transformation is recommended on the basis of this and a previous paper.

Use of tissue culture in predictive testing of drug sensitivity in human ovarian cancer. Correlation between in vitro results and the response in vivo.

The present work uses an in vitro test model to measure the sensitivity of human ovarian cancer cells to Melphalan and Melphalan combined with Adriamycin. With this model we studied the possible correlation between the in vitro results and the response to these cytostatic drugs in vivo. Cancer cells from 20 patients with advanced ovarian cancer were tested. The in vitro effects of the drugs were measured as differences in incorporation of labeled 3H-thymidine in drug containing tubes and in control tubes. The effects of the drugs on the different cancer cells varied greatly from strong sensitivity to resistance. In vitro and in vivo results were compared one year after start of patient treatment. The overall agreement was 16/20, 80%. The in vitro method thus estimates a tumor cell characteristic which has a biological meaning also in in vivo conditions.

In vivo suppression of Ehrlich ascites carcinoma by human antibody-dependent leukocytes.

Ceruloplasmin in Hodgkin's disease.

In 56 patients with Hodgkin's disease different clinical and histological stages the activity of polyphenoloxidase was repeatedly examined in the course of the disease as an indicator of serum ceruloplasmin concentration. In untreated patients the ceruloplasmin concentration was on average twice as high as in healthy controls. Repeated examinations made in the course of the disease demonstrated a good correlation of the values with the clinical condition of the patient, with the exception of some terminal stages and conditions following repeated chemotherapy, when liver damage was expected. The decrease to control levels in the course of therapy indicated the onset of remission. In contrast, increased levels indicated progression of the disease and necessity of treatment prolongation. Excluding intercurrent infections, higher ceruloplasmin levels above those found at remission were a clear sign of the progression. The correlation of ceruloplasmin values and erythrocyte sedimentation rate (1 h) was highly significant. The diagnostic and prognostic value of ceruloplasmin and/or serum copper determination is discussed.

Non-invasive methods for detection of cardiomyopathy in the course of antineoplastic treatment.

In our group of patients suffering from cancer and treated by polychemotherapy and monitored in a complex non-invasive way we obtained data as follows: A normal physical, sensoric and biochemical cardiac finding prior to treatment has been observed in 47 patients out of 68 (69.1%). A pathological cardiac finding prior to treatment has been found in 18 patients out of 68 (26.5%). Pathological changes evidencing for cardiomyopathy (CMP) induced by chemotherapy have been detected in 19 patients out of 68 (27.6%). Out of 18 patients with a pathological report prior to treatment in 8 of them the polycardiographic, echocardiographic, and biochemical alterations deteriorated during the chemotherapy. CMP induced by polychemotherapy has been found altogether in 27 patients out of 68 (39.6%). In this group of patients with clinically and biochemically detected CMP due to antineoplastic treatment, we found a significant decrease of ejection fraction (EF) (values less than 50%) and a marked increase Weisler's index (WI) (values greater than 0.520) approximately in 30% of patients. Our preliminary results show that polycardiographic examinations of systolic time interval (STI), the EF detected echocardiographically, as well as determination of myocardial isoenzyme creatine phosphokinase (CK-MB) activity represent useful non-invasive methods of early detection of any myocardial damage in the course of actinochemotherapy. A systematic monitoring of patients reduces the uncertainty with antitumor chemotherapy so that it enables--in a majority of cases--to bring the planned antineoplastic treatment to a successful end.

Prediction of cancer incidence in Cracow and Bratislava.

Projections for the incidence of all and selected major sites of cancer in the cities of Cracow and Bratislava were constructed for each year of the period 1978-1982 on the basis of data observed in the years 1968-1977. More or less increasing of unchanging trends were predicted for the malignant neoplasms of bladder, larynx and lung in males and of breast and ovaries together with decreasing trends of uterine cervix in females in the both cities. The trends predicted for the cancer of stomach in both sexes, of prostate and uterine corpus as well as of all sites in males showed different orientations in the compared cities. The factors connected with and usefulness of the adequate predictions of cancer incidence are discussed.

Potentiating effects of laser radiation on some immunological traits.

In vitro laser irradiation of peripheral blood lymphocytes increased the activity of T and B lymphocytes, determined by active rosette-forming cells and by immunofluorescence. Time and dose-dependent of irradiation was studied. The increase of lymphocyte activity was found, both in normal controls and in cancer patients.

Radiosensitizing ability and cytotoxicity of some 5(4)-substituted 2-methyl-4(5)-nitroimidazoles.

Radiosensitizing efficiency and cytotoxicity of three 2-methyl-4(5)-nitroimidazoles substituted with electron-withdrawal groups -NO2, -Cl and -OCH3 in 5(4)-position ("ortho" position) have been evaluated in vitro on V 79-379 A cells under aerobic as well as under hypoxic conditions. It has been established that 2-h incubation of cells at 37 degrees C in the presence of increasing (up to 2 mM) concentration of the compounds causes a moderate cytotoxicity for P7(2-methyl-5(4)-chloro-4(5)-nitroimidazole) and P5(2-methyl-4,5-dinitroimidazole). Cytotoxicities of these compounds are much stronger under hypoxic than under aerobic conditions. The compound P9(2-methyl-5(4)-methoxy-4(5)-nitroimidazole) is practically completely nontoxic. The radiosensitizing efficiency of P5, P7, and P9 at 2 mM concentration expressed by ER (enhancement ratio) equals 1.75, 1.57 and 1.14 respectively. A discussion regarding the features of electron-affinity, cytotoxicity and radiosensitizing ability of the compounds studied is presented.

Chromosome aberration persisting in cells of continuously irradiated mice.

Cytogenetical analysis of the bone marrow cells in mice irradiated continuously with the dose rate 0.478 Gy/day made within the course of various accumulated doses demonstrated the decrease in the number of chromosome aberrations till the post irradiation day 3 in all groups reaching about 75% on average. At further examination periods on day 10 and 30 the decrease in the aberrations appeared to be slower and was dependent on the magnitude of the accumulated dose. Increased number of the aberrant cells as compared with the controls was noted in the groups with the accumulated dose higher than 10 Gy and on the hundred postirradiation day, as well.

Metastasis of a cultured human bladder carcinoma cell line transplanted into nude athymic mice.

Earlier reports concerning metastasis from human tumors transplanted to nude mice indicate that the metastatic potential is correlated with invasive subcutaneous growth. It also seems that cultured cell lines are more prone to metastasis than heterotransplanted solid tumors. A malignant human uroepithelial tumor, grown in culture since 1970, was injected subcutaneously to six nude mice. All animals developed locally invasive tumor nodules. Postmortem examination revealed metastatic growth in the pancreas, in the mesenteric fat tissue, in the stomach wall and intestines and in the lymphatic vessels of mesentery. The findings are well in accordance with earlier reports.

Cytogenetical changes and their relation to survival of bone marrow hemopoietic cells during continuous irradiation.

In this paper the frequency of aberrant cells in the bone marrow of continuously irradiated mice with the dose rates 0.05 Gy/day and 0.50 Gy/day was studied. The aberrant cell values were found to be increased non-proportionally with the accumulated doses and their level was influenced mainly by the irradiation period in the dose range 0.5-5 Gy. The most significant reduction in the lymphocyte number and to some extent the granuloid cells number in the bone marrow occurred in the dose rate 0.50 Gy/day. The frequency of aberrant cells in the bone marrow increased at the same time to 15-20%. Possible mechanisms influencing the different response of cells to irradiation are discussed.

Clinical factors related to the presence of estrogen receptors in breast cancer: a prognostic stratification analysis.

The techniques used for determination of hormone receptors have provided a biochemical basis to the concept of hormone-dependence in breast cancer. By medical experience clinical factors were established for determination of hormonal treatment in breast tumors. The criteria used need now to be re-evaluated in terms of hormone receptor contents. We analysed a group of 155 patients with histologically confirmed breast carcinoma in which estradiol receptor determination was performed. Of these tumors 54% had more than 10 fmol of estradiol per mg protein. Menopause, advanced age, late first childbirth, obesity, early menopause and nuliparity were associated, in that order, with the presence of estrogen receptors in breast tumors. Prognostic stratification analysis brought out polar groups of estrogen receptor contents. Young, premenopausal women with children had only 16% estrogen receptor-positive tumors. Postmenopausal women, either without children or with late first childbirth had as much as 81% of estrogen receptor containing tumors. Clinical variables related to the presence of estrogen receptor positive tumors in the breast cancer population were coincident to risk factors for breast cancer in the healthy population. Possible implications of this coincidences are discussed.

CEA levels in active and non-active malignant tumors.

A total of 895 CEA estimations were carried out on serum samples from 283 patients with different primary tumor sites. All assays were performed during the period of follow up or treatment of the patients. Concomitantly with CEA estimation, activity of disease was recorded, based on objective laboratory and clinical parameters. The results obtained proved a high accuracy rate for normal CEA values in non-active disease as well as elevated CEA values in active disease. It is concluded that the correlation between CEA level and activity of disease in various neoplasms in an important aid in clinical decision making during the course of disease, provided false positive or negative results are taken into consideration.

Interrelation between lipidemia and somatomedin activity in cancer and age-associated pathology.

The study included 191 patients with obesity, atherosclerosis, diabetes mellitus, endometrial cancer, breast cancer and healthy subjects of various age. Somatomedin activity was determined by incorporation of radioactive natrium sulfate in vitro into the cartilage of female rats. The results of the study showed that somatomedin activity was not changed in subjects with normal metabolic parameters and ranged from 0.47 to 0.69 U/ml. In patients with diabetes mellitus, atherosclerosis and obesity accompanied by increased blood concentration of cholesterol and triglycerides, somatomedin activity rose up to 1.36- 1.62 U/ml. In patients with breast and endometrial cancer somatomedin activity was also increased, particularly in those with hypercholesterolemia and hypertriglyceridemia (3.04 U/ml for breast cancer patients and 2.20 U/ml for endometrial cancer patients). Theoretically, this may promote proliferation of somatic cells and thus contribute to tumor processes in oncological patients whose pool of cells is extremely sensitive to mitogenic agents.

Maturation index values and estradiol blood levels in uterine cervical cancer.

Maturation index (M.I.) values and blood estradiol-17-beta levels were estimated in a series of menopausal uterine cervical cancer patients and non cancer healthy menopausal women. Blood estradiol-17-beta levels did not exhibit differences in between the studied groups of cases. Three groups of uterine cervix cancer patients (before treatment, 6 weeks after radiation and 2 and more years after treatment) are all characterized by different M.I. values representing entirely different type of menopausal cytology. Atrophic type before treatment; "mixed" type 6 weeks after radiation and proliferative-estrogen type in 2 and more years after treatment. Very high karyopyknotic index values (KPI) in the group before treatment and very low values in the late after treatment group seem to support some scepticism in regard to strict specificity of M.I. and KPI as for expressing estrogen activity only.

Prognostic significance of reactive changes in regional lymph nodes in gastric and mammary carcinomas.

Regional lymph nodes in 80 cases of gastric and 70 cases of breast cancer have been examined and the reactive types have been correlated to the survival periods of the patients. In both groups of patients, a prognostically favorable sign was the detection of lymphocytic predominance combined with sinus histiocytosis, moreover, in patients with gastric cancer also the reactive type of predominance of germinal centers with sinus histiocytosis. Prognostically unfavorable sign was the detection of lymphocytic depletion, in particular if sinus histiocytosis was absent. The presence of metastases in the lymph nodes--apart from the reactive type--cannot be regarded as a sign of bad prognosis.

Hairy-cell leukemia and toxoplasmosis.

Investigation for Toxoplasma gondii infection using complement fixation test and microprecipitation method in agar gel was performed in fifteen patients with clinically and morphologically typical hairy-cell leukemia. Positive complement fixation test was found in four patients. In three patients an initially high complement fixation titer or its considerable increase associated with positive microprecipitation in agar gel suggested a recent toxoplasmosis. The importance of search for Toxoplasma gondii infection in hairy-cell leukemia patients especially before splenectomy and the necessity of reinvestigation after splenectomy is stressed.

Modified megathrombocyte index in the diagnosis of thrombopoiesis injury produced by cancer chemotherapy.

The border-line separating the normal platelet from the megathrombocyte was lowered from 2.5 microns, classically used, to 2 microns. This modification allowed more precise definition of normal frequency of megathrombocytes among platelets (megathrombocyte index--MI) which was 10-35%. The modified MI determination demonstrated the same kinetics of changes following the intensive cancer chemotherapy as the classical one and, moreover, enabled individual diagnosis of thrombopoiesis perturbation induced by chemotherapy which was not possible with the classical MI.

Oxygen tension and prediction of the radiation response. Polarographic study in human breast cancer.

Serial polarographic measurement of the tissue oxygen tension (pO2) was made in the course of fractionated irradiation (preoperative or sole treatment) of advanced breast cancer in 24 patients. In responsive tumors increase in pO2 appeared sooner before expressive tumor size reduction became noticeable. Repeated recording of unchanged pO2 values has proved to be a good prognostic indicator of local failure. The study made on this tumor model has shown that serial polarographic pO2 determinations with suitable electrodes causing minimal trauma and providing consistent and reproductive data about changes in tumor microcirculation and oxygenation, may enlarge the scale of indicators of the radiation response.

Inducible nature of tolerance to lesions produced by ultraviolet light in DNA of Chinese hamster V79 cells.

Chinese hamster V79 cells irradiated by split doses of ultraviolet light (2.5 + 5.0 J m-2) synthesize higher molecular weight DNA molecules than those irradiated by a single dose (5.0 or 7.5 J m-2). A second dose applied 8 h following the first one results in a higher portion of higher molecular weight molecules both in relative and absolute terms. Molecular weight of DNA pulse-labeled 8 h after a dose of 2.5 J m-2 is moderately higher than that of pulse-labeled DNA in unirradiated cells, even after the DNA synthesis in preirradiated cells has been fully recovered. Since the amount of DNA synthesized during the 8 h time interval between the two doses is not lower than that of DNA synthesized during an equal period in unirradiated cells, the synthesis of higher molecular weight molecules is not likely to represent elongation of part of the replicons, the other part being blocked. It is supposed that the induction produced by the first dose is involved in the replication of the damaged template.

Correlations between quantum chemical indices and carcinogenicity of methylbenz(a)anthracenes, methylbenzacridines and their model metabolites.

Carcinogenic activity of methyl derivatives of benz(a)anthracene and benzacridines has been correlated with the indices of electronic structure representing the reactivity and lipophilic coefficients of the compounds. The effect of the enzyme oxidase on the compounds has been stimulated and considered in correlations. The correlations with carcinogenic activity have been shown to be important from the statistical point of view: 1. correlation of the indices representing the lipophilic coefficient of the original compounds; 2. correlation of HOMO energies representing ease of formation of radical cations; 3. correlation of the reactivity indices in the A-region in the model complex of the studied derivatives and enzyme; 4. correlation of the reactivity indices of ultimate "carbonium" triol. Of these correlation equations only the equation 3 has shown wider applicability (i. e. also for benzacridines).

Cytofluorescence evidence of adriamycin in liver.

Fluorescence study was carried out in Sprague-Dawley rats injected intravenously with the anti-cancer drug adriamycin (ADR) at a dose of 15 mg/kg to determine the site of its localization in liver. Significant fluorescence emission of ADR nuclear-binding appeared in the liver shortly after the drug administration. The occurrence of the nuclear fluorescence in our material was explained by the binding of ADR to DNA. The cytoplasmic drug loading of liver cells might reflect their role in the drug metabolism.

Comparative pulmonary tumorigenesis in DBA/2J and C57Bl/6J mice by administration of 3-methylcholanthrene and dimethylnitrosamine singly and combined.

C57Bl/6J mice, which are inducible for both hepatic and pulmonary aryl hydrocarbon hydroxylase (AHH), and DBA/2J mice, which are noninducible for hepatic AHH but moderately inducible for pulmonary AHH, received dimethylnitrosamine (DMN) i. p., or methylcholanthrene (MCA) orally, or a combination of both agents, for 10 weeks; the animals were observed for an additional 26 weeks. The dosing schedule and total dose of DMN or MCA or DMN + MCA received were identical to those used by other investigators in their syncarcinogenesis bioassay study in Swiss mice. The lung lesions induced in the present experiments were alveologenic tumors and adenomatosis. Alveologenic tumors were induced in a much larger number of DBA/2J mice than in C57Bl/6J mice (87.9% versus 14.0%). If, however, the sum of alveologenic tumors and adenomatous nodules is considered and is expressed per lung lesion bearing mouse, then these ratios are for the control, MCA, DMN and MCA + DMN groups: 2.7, 1.5, 3.5, and 5.4 in the DBA/2J mice and 1.0, 1.3, 2.7, and 3.6 in the C57Bl/6J mice, respectively. This suggests a relatively greater susceptibility of the C57Bl/6J strain if the ratios are compared to the respective control values. This greater susceptibility of the C57Bl/6J is best seen by comparing the percent increase of the ratio for the MCA + DMN groups; the net increase is 100% for DBA/2J and 260% for C57Bl/6J. In the DBA/2J strain more extrapulmonary tumors (hemangioendotheliomas, liver and kidney tumors) were induced than in the C57Bl/6J strain by DMN or MCA + DMN, but not by MCA alone.

Thioproline: an inhibitor of chemical carcinogenesis.

Experimental DBA/1 mice were implanted with methylcholanthrene impregnated filterpaper disks. The disks were removed six weeks after implantation and median induction of fibrosarcomas occurred in 114.5 +/- 6 days. Two weeks pretreatment with thioproline, a reverse transformation drug, followed by maintenance throughout the duration of the experiment (27 weeks) reduced the tumor incidence to 60% that of controls. Pretreatment with thioproline and drug administration continued only through the six weeks post-implantation period failed to alter tumor incidence.

Further study of specific immunoreactivity in patients with malignant melanoma of uveal tissue by LAI assay.

Cell immunity was followed in patients with malignant melanoma of the uveal tissue with the aid of LAI (leukocyte adherence inhibition assay). The LAI test proved positive in 59 out of 65 patients with histological evidence of malignancy of the melanoma (90.7%). The test was repeated three to six months following excision of the tumor from the eye in 48 patients and in 19 of them (39.6%) it was found to be negative. All these patients have survived and after a mean lapse of 2.5 years following the surgical intervention no metastases have been observed. As against this, LAI positivity persisted in the remaining 29 patients (60.4%), with metastases present in the liver or the eyeball in 8 of them (27.6%) during the follow-up period (1-4 yrs). Out of 39 patients with the diagnosis of suspected malignant melanoma of the uveal tissue, the LAI test proved positive in 17 (43.6%), but as subsequently ascertained, 6 of these (15.4%) had benign choroid nevi. In 6 further patients with suspected malignant melanoma of the uvea in whom the LAI test had been negative, this disease was subsequently ruled out. From a control group of 101 patients with various non-tumorous diseases of the eyes, a false positivity of the LAI test was noted in only two (1.98%).

In vitro cell-mediated immune reactivity in colon cancer patients.

147 colon and/or rectum cancer patients in all clinical stages (according to TNM classification) aged 34 to 71 years were studied before radical surgery and 14, 45 and 90 days after it. The in vitro cell-mediated immunity was evaluated using the blastogenic transformation of blood lymphocytes to phytohemagglutinin (PHA), fast and total E rosettes. The immunosuppression observed in all clinical stages in comparison to donors becomes deeper 14 days after operation. 45 and 90 days after operation all three parameters studied increase but only in the clinical stage II (localized disease), the values reach and even surpass their initial level. Based on the obtained data conclusions were made in regard to the correlation of the lymphocyte functions to the clinical stage. In the localized (disseminated) stages a good (bad) ability of the immune system to recover is suggested.

Longitudinal follow-up circulating immune complexes of the serum in patients with breast cancer.

The values of circulating immune complexes (CIC) in patients with breast cancer were followed for one year at 4-month intervals by means of the "PEGIKEM" test. From the total of 67 patients, 37 were treated surgically and a significant decrease in mean values of CIC occurred 8 months after the operation. 30 patients with a more advanced stage of the disease treated with radiotherapy and chemotherapy showed a decrease in mean CIC values already after 4 months but later on the CIC values increased again. No significant differences in the mean CIC values prior to treatment between individual clinical stages were found, only a certain tendency towards a more frequent occurrence of higher CIC values in more advanced stages was noted. Nor were the findings in lymph nodes reflected in the mean CIC values prior to treatment. Comparison of CIC values prior to treatment and the results of 2-hour blood sedimentation, revealed conformity in only 40% of the patients. In the other cases higher values of blood sedimentation were more frequent. The study of prognostic value of CIC after 3 and 5 years follow-up is in progress.

Proliferative activity and radiosensitivity of human tumors.

On surgery material and biopsies from human tumors of various types investigation of labeling index and growth fraction in vitro, cell survival and cell population kinetics in diffusion chambers and changes in the cell kinetic parameters after irradiation were performed. The analysis of results obtained using the diffusion chambers and in vitro system demonstrate variability in labeling index within tumors of various type, individual differences and variation within the same tumor. Significant variability of growth fraction in individual tumors was also noted, however, in many instances, the most of cells in tumor were not proliferating. The growth fraction in gliomas and tumors of the floor of mouth was greater in tumors with high labeling index, in patients with a short tumor history and in patients with the short nonrelapse period. In the same groups of tumors changes of labeling index growth fraction during radiotherapy were compared with the therapeutic response of tumors. It can be concluded that the initial parameters of proliferative activity (labeling index and growth fraction) did not determine the radiation response of tumors but the dynamics and the intensity of their changes during the treatment were more informative in this respect. Complete regression of tumor may be expected only with decrease of labeling index and growth fraction.

A benzylidene mannopyranoside derivative with antitumor activity in the spontaneous mammary tumor system.

Methyl 2,3,4,6-di-O-benzylidene alpha-D-mannopyranoside (MeDBMP) was synthesized to evaluate its potential as a cytotoxic benzaldehyde liberating agent. MeDBMP was found to cause significant reductions in the sizes of spontaneous mouse mammary carcinomas and to produce long term alterations in their growth characteristics. It was nontoxic at all doses examined and possessed no mutagenic properties in the Ames Salmonella/mammalian-microsome test.

Intensity of proliferation and sensitivity of experimental tumors to the 1-methyl-1-nitrosourea.

The kinetics of tumor growth and cell proliferation of eleven generations of spontaneous mammary carcinoma in F1(CBA2 X C57B1/6) mice was studied. Factors responsible for different sensitivity of tumors to 1-methyl-1-nitrosourea were analyzed. The process of tumor transplantation was accompanied by the reduction in latent period and the increase in the mean specific rate of tumor growth. The rise in tumor growth rate in consequent generations was associated with the increase of the number of DNA synthesizing cells. The fall in the intensity of the DNA synthesis was observed along with the tumor growth within one passage. The sensitivity of tumors to 1-methyl-1-nitrosourea was correlated reciprocally only with the initial labeling index of tumors and the specific rate of tumor growth.