Search

The retinoblastoma gene product mediates the interaction between transcriptional factors and cyclin-kinase complexes, which perform a regulatory and effector function in the process of cell division. The activity of the retinoblastoma gene product is regulated by phosphorylation, which results in the appearance of additional protein molecules migrating in the region of 105--116 kDa during electrophoresis. Stereochemical analysis has established a direct correspondence between the extent of phosphorylation of the retinoblastoma gene product and its electrophoretic mobility. The results obtained permit an estimate of the phosphorylation of this protein in cells of different tissues by immunoblotting analysis of their lysates. The results of this study demonstrate that the degree of phosphorylation of retinoblastoma gene product in a series of stable cell lines increases as we go from monolayer to multilayer cultures, and further to cell cultures in suspension. Human cells produce more phosphorylated proteins compared to homologous mouse cells. The phosphorylation pattern of retinoblastoma gene product is probably tissue-specific. Much like mouse fibroblasts, HeLa cells may contain a hypophosphorylated protein with a molecular weight of 105 kDa. Phosphorylation of the retinoblastoma gene product in cells of embryonic mouse adenocarcinoma (line p19) changes in response to cloning or stimulation of differentiation by retinoic acid. The formation of all forms of retinoblastoma gene product, which pre-exist during asynchronous growth, is increased in the course of cloning. Differentiation is associated with the synthesis of an increased amount of hypophosphorylated protein. The results obtained lead to the hypothesis that even though the phosphorylation pattern of the retinoblastoma gene product is tissue-specific, it can show significant variation under the conditions of either cloning or differentiation and can be maintained for extended period of time at a new level that was not characteristic of the initial cell population.

Phosphorylation and dephosphorylation of the retinoblastoma gene product (pRB) are recognized as necessary events in the cell cycle progression. To study the role of pRB in regulation of cell proliferation, the stable cell lines with constitutive expression of the exogenous RB gene can be employed. In order to obtain such cell lines in this work C3H10T1/2 mouse fibroblasts were infected with defective retrovirus encompassing the RB and Neo gene conferring resistance to geniticine (G418). The pRB production and its phosphorylation pattern were analyzed by immunoblotting in cell lysates considering well known data on correlation between pRB phosphorylation pattern and its electrophoretic mobility. Cell lines subjected to G418 selection with the following cloning procedure were identical to the control cells expressing beta-galactosidase, when compared for pRB production and phosphorylation in the cell cycle stages characterized by hyperphosphorylated pRB. However, cells of the experimental cell lines hypophosphorylated pRB much faster and accumulated much more underphosphorylated protein compared to the control cell lines. The doubling time of the cells was not affected either by changes in the pRB phosphorylation pattern or by its overproduction during separate cell cycle stages. These results suggest that maintaining of the physiological level of pRB phosphorylation in cycling cells is strictly controlled and is considered to be a more important condition of the cell cycle progression than pRB dephosphorylation.

It is tried to differentiate between various stages of malignant evolutive push and normal mammary tumors to establish an individual therapeutic procedure. Besides mammography and thermometry, investigations of urinary excretions of estrogenic and androgenic steriods are qualified for this purpose. Patients with malignant evolutive push showed an increased excretion of estrogens which reached its highest levels in patients suffering from inflammatory mammary carcinomas. The excretion of 17-ketosteroids shows a behaviour vice versa. Altogether mammary tumors with malignant evolutive push are characterized by a state of absolute and relative hyperestrogenity respectively. This phenomenon preferably depends on the clinical stage of the disease than on the age of the patients.

Fractionated 17-ketosteroids were determined in 60 females with hirsutism. The chromatographic method was adapted in the Oncological Institute of Cluj-Napoca. In 32 patients the idiopathic form of hirsutism was confirmed with normal results for all the fractions and only sometimes a slight rise of androsterone. In 18 patients an ovarian origin was affirmed; all of them had androsterone and etiocholanolone over normal limits. In ten patients, who had an adrenal origin of hirsutism the great values of DHEA (dehydroepiandrosterone) were observed. In one patient with virilizing adrenal tumour, both total 17-ketosteroids, and all fractions were much elevated DHEA being over 100 mg/24 hours.

The main endocrine features in a group of 87 patients with inflammatory breast carcinoma with very rapid evolution (group I) and those of 400 patients with breast cancer with no inflammatory signs (group II) have been compared. The mean age at menarche showed a significantly higher degree in group I, where the later menarche was reported in 36.8% of cases (greater than or less than 16 years). The mean number of abortions has been decreased in the same groups. No significant differences regarding to parity, lactation and mean age of the menopause have been found in these two groups. The mean values for total urinary estrogens were significantly higher in group I. The main fractions of 17-ketosteroids were estimated in 60 women of group I and 61 women of group II. The mean levels of these hormones were more decreased in group I, the most significantly differences in these two groups were registered for ethiocholanolone. In the most cases with decreased levels of ethiocholanolone, the survival time was also shorter. Such data showed a more prominent endocrine imbalance in the inflammatory breast carcinoma in comparison with breast cancer patients of normal evolution.

The main fractions of 17-ketosteroids were estimated in 102 women with breast cancer. Sixty two patients were cases with rapid evolution (malignant evolutive push) or very rapid evolution (inflammatory breast carcinoma). The mean ethiocholanolone levels and partially androsterone levels were significantly decreased in patients with rapid evolution in comparison with the corresponding values found in patients with breast cancer of slow growth.

The main fractions of 17-ketosteroids were estimated in 30 young women (under 40 years) with breast cancer and 20 controls of the same age. The determination of the fractionated ketosteroids was made by thin layer chromatography on silicagel. The mean levels of etiocholanolone, androsterone, total ketosteroids and the sum of the fractions of 17-ketosteroids were significantly decreased in patients with breast cancer in comparison with the controls. No significant differences between the mean levels of dehydroepiandrosterone and 11-oxygenated-17-ketosteroids were noted in these groups.

Urinary outpout of oestrogens and 17-ketosteroids were estimated in 200 women with breast cancer; 60 of them were cases with rapid evolution (malignant evolutive push). The majority of cases analysed had abnormal hormonal levels, especially of oestrogens. The average oestrogenic levels are higher than the normal values; the highest values correspond to the most severe and rapidly evolutive type of breast cancer, the inflammatory breast carcinoma. In the patients without evolutive push, the proportion of cases with absolute or relative hypooestrogeny is approximately equal toor exceeds the proportion of hyperoestrogenic cases. In the presence of the evolutive push and especially of his most severe type, inflammatory carcinoma, the cases with hyperoestrogeny, both absolute and relative, by hypoandrogeny, are dominant. The proportion of cases with higher 17-ketosteroid urinary outputs than the normal mean is greater in the patients without evolutive push, while in those with evolutive push, the majority of cases have lower values of 17-ketosteroids than the normal mean.