Concanavalin A receptors on the surfaces of human breast cancer cells in organ culture.

The binding of concanavalin A (Con A) at the free apical membranes of surface tumor cells in human breast cancer explants grown in organ culture was studied cytochemically with horseradish peroxidase (HRP) as a marker. On the cell membranes in aldehyde-fixed explants or explants exposed to Con A at 4 degrees C, a continuous label covered the entire free surface of the cell, which indicated the dispersed distribution of Con A binding sites. The binding of Con A at 20 degrees C resulted in discontinuous label of the cell surface, with gaps of unlabeleled membrane and partial endocytosis of the label. Incubation at 37 degrees C, following the binding of Con A and HRP at both temperatures, induced more extensive, incubation time-dependent discontinuities of the surface label that led to complete disappearance of the label from the surface and its eventual endocytosis. Con A was topically stablized on the surface only in those regions where two membranes or a different part of a folded membrane were in close contact. No differences were found in the binding, redistribution, and internalization of bound Con A among the various tumors studied.

Tumor progression of murine epidermal cells after treatment in vitro with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid.

Tumor-promoting or antipromoting agents potentially may act directly on initiated squamous epithelial cells or indirectly through effects on normal keratinocytes or immune cells. The purpose of this study was to examine direct effects by comparing in vitro and in vivo treatment of initiated cell populations with 12-O-tetradecanoylphorbol-13-acetate (TPA) or retinoic acid. Keratinocytes were initiated by treatment in vitro with 7,12-dimethylbenz[alpha]anthracene. Replicate cultures of a cloned initiated cell line were exposed to TPA or retinoic acid with acetone as control. After an equivalent number of population doublings, cultured cell sheets were transplanted as skin grafts to athymic nude mice. Replicate grafts from each in vitro treatment group were then treated with TPA or retinoic acid for 8 months. Promotion was quantified by tumor incidence (graft sites with tumor per total sites) and by tumor growth rate. The findings were as follows: (a) TPA increased tumor incidence whether it was applied in vitro or in vivo; (b) TPA in vitro favored more progressive tumors than TPA in vivo; (c) stages of malignant progression from cloned keratinocytes treated in vitro were histologically identical to those following treatment of skin in vivo, including papilloma, dysplastic invasive papilloma, squamous cell carcinoma, and metastasis to lymph node and lung; (d) retinoic acid treatment in vivo reduced tumor incidence and tumor growth rate in initiated cells previously exposed to TPA but not in cells previously exposed to retinoic acid. The results indicated the following: (a) direct effects of TPA on initiated keratinocyte populations were a significant component of tumor promotion; (b) factors in vivo modified the TPA response toward less progressive growth; and (c) the effect of retinoic acid was modulated by prior treatment history.

Variation of vascular density within and between tumors of the uterine cervix and its predictive value for radiotherapy.

Vascular density in different regions of a number of uterine cervix carcinomas was determined by morphometric analysis of stained histologic sections. Variance analysis indicated a larger inter- than intra-tumoral inhomogeneity of the vasculature, suggesting a certain individual vascularization pattern in these tumors. As indicated by a retrospective study of archival biopsies, this pattern has a predictive value in regard to the efficacy of radiotherapy.

Cytotoxic interactions of Zn2+ in vitro: melanoma cells are more susceptible than melanocytes.

Previous studies have shown that sensitivity to high extracellular levels of Zn2+ is a general feature of cells in vitro and that a prerequisite of the toxic action of zinc is entry into cells via channels that are shared with iron or calcium. As the biochemical and toxicological behaviour of zinc chelate complexes could be different from that of free Zn2+, the effect of chelating agents on zinc transport into human melanoma cell lines was tested. EDTAcal and tetracycline reduced the toxic action of zinc ions in vitro, whereas phenytoin and diethyldithiocarbamate potentiated its effects. D-penicillamine, an effective chelator of zinc in vivo, also exerted a protective action in vitro. Comparison of sensitivity to Zn2+ in vitro between human melanoma lines and several lines of pigment cells from skin of various origins demonstrated that melanoma cells are killed by zinc ions at concentrations which are only partially toxic for normal pigment cells. This is consistent with the repeatedly observed high uptake of 65Zn by melanoma cells.

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.

Colony growth in soft agar of human melanoma cells from biopsy material, cell lines and xenografts.

Colony growth in soft agar of human melanoma cells from biopsy material, cell lines and xenografts was evaluated. Colony forming potential is constantly very low in all studied types of tumor tissue, however slight increase in clonogenic potential was seen during subsequent xenograft passages, but within the range of less than 1%. Results of presented studies suggest that the number of colonies does necessarily express the degree of colony forming potential and the culture preparation and conditions as well. It appears that there is no reliable correlation between the number of colonies in soft agar and the clinical course of melanoma patients under study.

Estrogenic effect in vaginal smears in cancer of the uterine cervix.

Values of karyopyknotic index (KPI) in a series of 37 uterine cervix cancer patients were significantly increased when compared with the same groups of 61 healthy women. Radiosensitivity of the tumors was accompanied by subsequent decreasing in the KPI values while in all radioresistent cases as well as in all early recurrences persistence of initial high KPI values could be proved. Significant differences in the kinetics of KPI changes during and after radiation in the group of praemenopausal and postmenopausal patients support suggestion for non ovarian origin of this estrogen like activity.

PIP: Estrogenic effect in vaginal smears in cancer of the uterine cervix was studied by determining values of karyopyknotic index (KPI) in 37 patients and 61 healthy control women. Vaginal smears were stained by the Papanicolaou method and KPIs were estimated by counting 300-400 superficial squamous cells. The results indicated that KPI values in uterine cervix cancer patients were nearly 3 times higher in all age groups when compared with the same age groups of healthy women. Radiosensitivity as expressed by local healing disappearance of the tumor was accompanied by subsequent decreasing of the KPI values, while in all radioresistent cases as well as in all early recurrences, persistence of initial high KPI values could be proved. A decreasing trend of the KPI values during and after radiotherapy was more expressive and immediate in premenopausal patients. Postmenopausal patients were characterized by less increased initial KPI values, which supports the suggestion for nonovarian origin of this estrogen-like activity.

Chromosomal aberrations, sister chromatid exchange (SCEs) and micronuclei induction with three platinum compounds (cis-DDP, CHIP, CBDCA) in V79 cells in vitro.

Three platinum compounds (cis-DDP, CHIP, CBDCA) were tested or induction of chromosome aberrations and micronuclei as well as for SCEs in V79 cells in vitro. There was a clear relationship between the degree of inhibitory activity of the tested drugs and the increase of studied markers of cytogenetic damage. Statistical significance was proved in increase of SCEs values in cis-DDP and CHIP-treated cells when compared with untreated controls.

Changes in distribution of the phases of mitosis in human tumor xenografts following drug treatment.

Distribution of individual mitotic phases in different human xenografts on nude mice showed significant changes after treatment with a range of drugs when compared with untreated controls. The total amount of mitoses in prophase significantly decreased in all responsive tumors while those in all other phases increased but in a very variable degree. There was some correlation in between the range of the mitotic phase distribution changes and the degree of tumor response to treatment.

Relationship between melanogenesis, proliferative activity and response to chemotherapy of human melanoma xenografts.

Wide range of different drugs used in the cytotoxicity studies in an amelanotic human melanoma xenograft on nude mice showed a clear inverse relationship between melanogenesis and proliferation. Decreasing rates in proliferative activity estimated by mitotic answer expressed by phase distribution changes as well as by the tumor volume response to drug treatment were in direct relationship with increasing rates in the degree of melanogenesis expressed in percentage of melanin containing cells in histological specimens.

Flow cytometric analysis of primary cervical and vulvar carcinomas and their metastases.

In a series of 25 patients with cervical carcinoma and 35 patients with vulvar carcinoma, clinically classified (FIGO) as Stage I and Ib, respectively, DNA ploidy and S-phase cell fraction were estimated in paraffin-embedded samples of the primary tumors and their metastases by means of flow cytometry (FCM). The two groups of patients were selected cases in whom lymph nodes removed at radical operation were histopathologically verified as metastatic ones. Prevailing part of primary tumors of both anatomic sites had diploid DNA content and low S-phase fraction. Irrespective of the DNA content and low proliferative activity the early stage carcinomas behaved aggressively as indicated by the presence of metastases, the DNA content of which corresponded in most of the cases with that of primary tumor. It appears that prediction of the tumor growth based only on cytogenetic and cytokinetic parameters may be connected with difficulties.

Stimulation of differentiation in human melanoma cells by dimethyl sulphoxide (DMSO).

Stimulation of differentiation in human amelanotic melanoma cell line by 0.5, 1, 1.5 and 2% DMSO expressed in increased melanization was linked to significant decrease in proliferation rate of these cells. After removing DMSO the 0.5 and 1% samples became within 14 days again amelanotic with the same growth capacity as untreated controls. The 1.5 and 2% samples did not recover after DMSO removing and the inhibition of proliferation was irreversible.

The effect of anemia on the radiotherapy results of the uterine cervix cancer.

Relationship between hemoglobin level and survival and/or recurrence rate was studied in radiation treated group of postmenopausal women with uterine cervix carcinoma. The data suggest that anemia alters unfavorably the effectiveness of radiotherapy. A possible explanation of the lowered control rate is that due to deficiency of tissue oxygenation by reduced oxygen carriage proportion of hypoxic cells increases and impairs the radiosensitivity.

Flow cytometry (FCM) of early radiation response in epidermoid carcinoma of uterine cervix.

DNA flow cytometry (FCM) investigation of tumor specimens before and after 30 Gy 137Cs radiation treatment was performed in 33 cases of epidermoid uterine cervix carcinoma. Distinct differences in the type of FCM response to radiation were seen when the results of DNA index (DI) in diploid and aneuploid tumors and proliferation index (PI) values in diploid tumors from pretreatment and 30 Gy irradiated specimens were compared. We observed partial or total reduction of PI in 12 of 17 diploid and near diploid tumors, and total reduction of the aneuploid population in 14 of 16 aneuploid tumors. No significant correlation was found between the type of FCM response and clinical stage of the disease or the histological degree of differentiation.

Vascularization and radiocurability in cancer of the uterine cervix. A retrospective study.

The proportion of vascular elements in relation to the parenchymal and stromal components was determined by morphometric analysis of the histologic preparations from biopsies of uterine cervix cancers. In all cases, the material was obtained from tumors in Stages Ib and IIa before any therapy was initiated. In the 23 cases in which radiation treatment of the lesions resulted in a survival longer than 5 years, the proportion of blood vessels in the neoplastic tissue was found to be larger, and especially the stromal components were richer in vascular elements than in the 22 cases in which survival was shorter than 5 years. It was concluded that vascular density at least in cervical cancers of the stages studied can be a diagnostic parameter of a prognostic value as well as of a therapeutic usefulness.

Flow cytometry (FCM) analysis of endometrial hyperplasia and carcinoma.

In a series of 52 biopsy specimens (31 endometrial carcinomas, 10 atypical endometrial hyperplasias, and 11 cases of normal endometrium), DNA ploidy and S-phase cell fraction were estimated in paraffin-embedded material. DNA aneuploidy was detected in 2 of the 10 atypical endometrial hyperplasias and 7 of the 31 endometrial carcinomas. The majority of aneuploidy was found to be connected with the loss of tumor differentiation. No ploidy disturbances were found in normal endometrium. The S-phase cell fraction value of normal endometrium was significantly lower when compared with that of endometrial carcinoma. The broad variation in S-phase cell fraction values of the endometrial carcinomas and atypical endometrial hyperplasias was in contrast with the low variability of S-phase cell values of normal endometrium. Very low incidence of aneuploidy in the group of well differentiated endometrial carcinomas (Grade I) enables the suggestion that the presence of aneuploidy predicts a more aggressive disease and that the detection of an aneuploid stemline in atypical endometrial hyperplasia may already indicate the neoplastic transformation.

Prognostic significance of vascular density and a malignancy grading in radiation treated uterine cervix carcinoma.

In a retrospective study of 95 patients with squamous cell carcinoma of the uterine cervix (Stage IB, IIA, III) treated by radiation only, pretreatment biopsy material was used for assessment of the prognostic value of histopathological multifactorial malignancy grading and a morphometric estimation of vascular density in stroma. By comparison of the two systems, vascular density has been proved superior to malignancy grading with respect to prognostic value.

Cell proliferation kinetics and nuclear morphology in endometrial cancer under progesteron treatment.

Cell proliferation kinetics by using the double labeling with 3HTdR--14CTdR, and nuclear morphology were studied in 20 patients with endometrial cancer who were given progesteron as a preliminary therapeutic measure. Results of these studies indicate that we have to face the fact that in all histological types of endometrial cancer considerable variations in the amount of tumor cells undergoing secretory conversion occur due to the primary heterogeneity of the tumor cell population.

Approaches to prove the melanoma origin in amelanotic human melanoma cell lines.

The human melanoma B-HM8 cell line was derived from highly pigmented malignant skin melanoma. After 5 weeks of cultivation it entirely lost the pigmentation and has remained amelanotic since. Electron microscopy revealed neither premelanosomes nor melanosomes and the cells did not release detectable amount of dopa-oxidase activity into culture medium. Immunocytochemical studies using the polyclonal anti-S-100 antibody and detection of alpha-mannosidase activity in culture medium proved the melanoma origin of B-HM8 cells. Chromosomal changes in the karyotype of these cells were typical for human melanoma with chromosomes No. 1, 5, 7, 9, and 11 involved most frequently.

Human melanoma cell lines: morphology, growth, and alpha-mannosidase characteristics.

Four human melanoma cell lines were characterized by evaluation of the morphology, ultrastructure, cytogenetics, plating efficiency, agar colony formation and alpha-mannosidase values. Biochemical studies demonstrate that all studied lines produced alpha-mannosidase even under condition of long-term in vitro growth. All respective lines were characterized by a great variations in biological markers studied and very few reliable comparisons could be obtained as regards the proliferation activity and capacity, cellular composition and clonogenic potential as well. Due to cellular selections during long-term in vitro cultivation, the range for using these experimental systems as models for the study of biology of human malignant melanoma seems to be rather limited.