Biomedical subjects
Publications and source records attributed to I N Shvemberger.
Transgenic mice are a suitable model for studying problems of modern biology on the molecular, cellular, and tissue levels. It is shown that some transgenic mice with integrated c-myc, v-myc, T-ag SV40, BPV have spontaneous tumours and may be used to study the role of oncogenes in the tumour development.
Six transplantable murine tumors of different histogenesis were investigated after transplantation to subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). Cell morphology was studied using light microscopy. DNA contents in the nuclei of tumor cells were investigated with flow cytometry technique. LDH isoenzymes were studied using electrophoresis in polyacrylamide gel. In the case of tumors with near diploid modal class, a redistribution of LDH isoenzyme activity and an increase in morphological differentiation level were obtained. In the case of tumors with modal class differing from the diploid one, a morphological structure changes were revealed, but there were no differences in LDH isoenzyme activity. The data obtained show that the capability of increasing morphological and biochemical differentiation level after cultivation in the EAC of murine transplantable tumors remains even on the late stages of progression in tumors of different histogenesis with near diploid value of modal class.
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The activity of lactate dehydrogenase (LDH) isoenzymes was studied in clones of rhabdomyosarcoma (RMS) MX-53, being transplanted on mice of CC57W line. Clones obtained in lungs after the intravenous injection of a tumor cell suspension were transplanted into subcutaneous connective tissue (SCT) and eye anterior chamber (EAC). A study of LDH isoenzymes spectrum in transplants and native muscle tissues has been made using polyacrylamide gel electrophoresis and densitometry. We revealed a variability of LDH isoenzyme activity within one clone, and in population of clones under different proliferation conditions. Under all the proliferation conditions, we observed spectra characteristic of tumors of a given histogenesis with predominating activity of M-subunits. But during proliferation in EAC the M:H ratio decreased, and LDH spectra of tumor cells became more similar to the LDH spectrum of normal skeletal muscles. It is concluded that intra- and interclonal variations of the character "LDH isozyme spectrum" is strongly influenced by the environmental factors.
Clones of rhabdomyosarcoma cells were obtained due to implantation of the tumor cells in the eye anterior chamber, in subcutaneous connective tissue and in lungs of mice. The DNA contents in the clone cells were measured using flow cytometry. Diploid indices of clones were calculated from the ratio of the modal content of DNA in G1-cells to the DNA content in lymphocytes contaminating these clones. The diploid indices of various clones varied from 1.4 to 2.3. The mean diploid index calculated for clones that grew under given conditions of proliferation varied within 1.85-1.88. Only one of the 66 clones examined displayed a two-peak distribution of cell according to their DNA content in phase G1, which may suggest a karyotypic instability of the progeny of the tumor clonogenic cell. No correlation was revealed between the diploid indices of clones and the following parameters, such as: the portion of tumor cells being at different phases of cell cycle; the number of normal (stromal) cells contaminating the clones, coefficients of variation of DNA contents in tumor cell clones. A positive correlation was observed between the coefficients of variation of DNA contents in the normal (stromal) cells contaminating the clones and those of tumor clone cells being in phase G1. It is concluded that the variability of results of the flow cytometric measurement of DNA in G1-cells may reflect the variability of cells in respect not only to their DNA contents but also to their capacity of dye sorbtion.
Four clone lines of transplantable cell polymorphic rhabdomyosarcoma A-7 were investigated during transplantation to the subcutaneous connective tissue (SCT) and into eye anterior chamber (EAC). Cell morphology of transplants was studied by light and electron microscopy, the activity of their LDH M- and H-subunits was examined cytochemically, and the quantity of their nuclear DNA--cytophotometrically. In the case of A-7/1, A-7/2 and A-7/3 cell lines of EAC transplants we noticed a decrease in cell element kataplasia levels, differences in LDH M- and H-form ratio, reduction in the karyotype variability. Transplants of A-7/4 clone line were similar in SCT and EAC for all the signs studied. The results obtained show that the transplantable cell polymorphic rhabdomyosarcoma A-7 is heterogeneous for its differentiation and normalizing capacities during EAC proliferation. The data reported elsewhere concerning capability of four lines of murine rhabdomyosarcomas to normalize in EAC are discussed, and some possible mechanisms of this effect are regarded.
The activity of LDH M- and H-forms, nuclear DNA contents and of genome mutation rates in the Ehrlich ascitic carcinoma cells were studied after transplantation to the intraperitoneal cavity, subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). SCT and EAC cultivations demonstrate the increase of M- to H-forms activity ratio due to a lesser H-form activity; so, LDH profiles appear to be associated with such a spontaneously highly differentiated adenocarcinoma of murine breast cancer. SCT and EAC cultivation leads also to changes in the karyotypic structure of cell population--there are no polyploid cells (DNA content is more than 4c). It may result from a sharp fall in genome mutation rates in the Ehrlich ascitic carcinoma clones following EAC proliferation which appears 47 times lower than those during lung proliferation. The data obtained favor a hypothesis that the increase in differentiation level and decrease in tumorigenety of cancer cells during EAC proliferation may be due to selection of the near-diploid cells and to the reduction in genome mutation rates, which in the whole results in decreasing genotypic and epigenotypic variability in tumor cell populations.
Tumors transplanted into the eye anterior chamber (EAC) were atypic, more differentiated morphologically and with higher activity of H-forms of LDH, compared to the same tumors grown in the subcutaneous connective tissue (SCT). Besides, in the EAC, the tumors were less karyotypically heterogeneous and had lesser mitotic indices, than in the SCT. After cultivation in the EAC, populations of tumor rhabdomyoblasts displayed a decreased transplantability in SCT, i.e. the usual site of their implantation. These results may suggest that the proliferation of tumor cell populations in the EAC, i.e. in the immunologically privileged place, can be accompanied by the reducing of their malignancy up to the total loss of the common tumorigenic ability. Even the cell populations of tumor rhabdomyoblasts from the late stages of tumor progression can undergo such a process of normalization.
Polymorphic CC57W mice rhabdomyosarcoma MC-53, after selection for malignancy, was investigated during the transplantation to subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). SCT and EAC transplants appeared to be equal both morphologically and ultrastructurally. Relationship between M- and H-forms of LDH, and the karyotype structure of tumor cell populations were invariably similar in SCT and EAC. However, after EAC proliferation, in contrast to SCT proliferation, transplantability of tumor rhabdomyoblasts decreased, which may be associated with a decrease in proliferative activity of tumor cells in the EAC. Our data allow to suppose that mouse rhabdomyosarcoma MC-53 cells, during selection for malignancy, lost its capacity of differentiating and normalizing in EAC, unlike the previously investigated rhabdomyosarcomas MC-62, MC-III and A-7. These results may be explained by the fast progression of this tumor line. It is obvious that capacity of tumor cells of differentiating and normalizing during the EAC proliferation may depend on the tumor histological type, on the retention capacity of tumor cell elements of differentiating and on the stage of progression.
A study was made of the transplantable rhabdomyosarcoma of mice M-62 during its transplantation after 15 passages simultaneously into the subcutaneous connective tissue and the eye anterior chamber (EAC), the latter being an immunologically advantageous site. The transplantable rhabdomyosarcoma cell populations, proliferating in EAC, are characterized by a higher differentiation level as compared with those proliferating in the subcutaneous connective tissue, the cells being taken from the same tumour, at the same passage. Morphologically it is expressed in the appearance of cytotypical (myofibrils in mononuclear tumour myoblasts) and histotypical (formation of myosymplasts) characters of differentiation. The increase in the intensity of differentiation correlates with changes in the ratios of M- and H-forms of LDH towards that which is characteristic of the definitive skeletal muscle tissue. A more intensive differentiation occurs on the background of changes in the karyotype structure of the populations of tumour myoblasts expressed in the increased share of diploid cells. An assumptions is made that the populations of tumour cells proliferating in EAC, i. e. under conditions of isolation from the immunological reactions of the organism, demonstrate changes towards normalization.
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The ultrastructure of rabdomyoblasts has been studied. Rhabdomyoblasts were removed from rhabdomyosarcoma M-38 induced in mice by 20-methylcholantren, from clon-line M-38/13 (5 passages) originating from this rhabdomyosarcoma, and from two line of rhabdomyosarcomas cultivated for several years. Throughout the passaging (progression) of tumors, both highly and poorly differentiated ((cambial) cells were seen to disappear from tumor cell populations. The intracellular differentiation of rhabdomyoblastse manifested itself as formation of specific contractile structures, can occur only in those rhabdomyoblasts which enter the cell complexes. These complexes have narrow intercellular spaces and intercellular contracts. It is suggested that the rhabdomyoblast with features of cytotypical differentiation may be considered as a structural functional unit of the differentiating cell complex.
Potentialities for evaluating the viability of the Zaidela ascitic hepatoma cells by the number of tumours formed after transplantation of cells to the rat's eye anterior chamber have been considered. Dose effect of far (254 nm) and near (300--380 nm) UV light on the cell tumoregenecity has been studied.
The study of rat testis seminoma by the method of clonal analysis and ectopic transplantations has shown that typical and anaplastic types are different stages of tumor progression in the course of which atypical and anaplastic features of the seminoma cell elements increase. It does not seem inconceivable that the spermacytic form, never encountered during our investigation of 400 tumors, may be an independent seminoma form incapable of transforming into a typical one. A spindle-cell form of seminoma has been described most frequently occurring during transplantation of a typical seminoma into testis and during cloning. The obtained evidence permits to consider the small dark cells of the seminoma as being cambial cells of the tumor which occur abundantly in growing tumors, and are absent in tumors with ceased proloferating. At later stages of the seminoma development, the small dark cells may be absent, their function being performed practically by all tumor cells. Acinar structures found in part of clones from lungs and in the majority of transplants from eye anterior chamber suggest a histogenetic relationship between the typical seminoma and embryocarcinoma, and thus enable us to regard the latter as the most differentiated variants of the seminoma.
A study has been made of 7 transplatable lines of mice rhabdomyosarcomas and one line of rat rhabdomyosarcoma during their transplantation into the eye anterior chamber subcutaneous tissue. In all, 10 subcutaneous transplants and 15 transplants into the eye anterior chamber (EAC) were examined. Etanol fixed print smears were subjected to the Feulgen reaction to measure the DNA content using a cytophotometer MCPhU-1; 100 cells being measured in each transplant. In the majority of the EAC transplants, a statistically significant decrease of the karyotypic variability was found in additionto the augmentation to the diploid cell ratio as compared to subcutaneously proliferating populations of the same tumour lines. In some cases EAC transplants displayed exclusively diploid (periploid) populations of tumour myoblasts. Shifts in the karyotypic structure of populations towards diploidy, revealed during the cultivation of transplantable rhabdomyosarcomas, may be regarded as a phenomenon of the "karyotypical normalization" of tumour cells. The disappearance or sharp decrease of tetraploid or hypertetraploid classes of cells in EAC transplants may be due to the increase of their selective value in condition of immunological privilege of diploid, karyotypically normal cells, and of reduction of the genome mutation frequency in a diploid fraction of tumor myoblast populations.
The anterior chamber of mammalian eye (ACE) is one of the places of organism privileged immunologically that permits to use it for transplantations of allogenic tissues. Besides, the medium filling the chamber, provides an excellent growth of transplants there, whereas the transparent cornea allows to control the growth. The analysis of results obtained with this method shows its acceptibility both for heterotransplantations of tumors including human tumors, and for homotransplantations with the aim to study the progression of tumors and the selection of constant tumor lines. The ACE is easily applicable for studying morphogenetic potentialities of tumor cells, as well as their ability to differentiation and normalization, because its medium has the quality of "differentiating" for many tissues. Cytological heterogeneity, revealed during tumor transplantations, allows to study the spectrum of cytological variability and transgression of tumor characters at different stages of their progression. The ACE can be used for estimation of tumor formation ability of tumor cells by determining TD50 (tumor dosis 50) which is important for estimation of different kinds of damage (X-rays, chemotherapy etc.). According to authors' point of view, it is very perspective to use the anterior chamber of eye for elaboration of effective methods of hybridization tumor cells with normal ones.
An electron microscope study was made of fibrillar structures in various types of cell elements in mice rhabdomyosarcoma. Different stages of myofibril development were followed in such differentiated cell elements as large spindle cells, large round cells and middle spindle cells. In the former two cells, the evolution has riched a stage of bundles of myofibrills with Z-like bands. The highest degree of myofibril maturation occurs in multinuclear myosymplasts. The occurrence of myofibrils being at different stages of evolution within the same cells is characteristic of tumor myoblasts in addition to an excentric unipolarity of cell differentiation and atypical structure of Z-brands. Cytotypical differentiation (myofibrillogenesis in uninuclear cells) may be the case only in those types of rhabdomyosarcoma cell elements which are able to undergo histottypical differentiation (fussion and formation of multinuclear myosymplasts).
12 mouse rhabdomyosarcomas from V--XI transplant generations were investigated at their growing in the anterior chamber of the eye, in the testis, lungs and subcutaneous fibrous tissue. With these modes of transplantation rhabdomyosarcomas were seen differentiating to different levels and differed in their cell populations. The most differentiated rhabdomyosarcomas were in the anterior chamber of the eye: they formed myotubes with cross-striated myofibrilles; the least differentiated ones occurring in lungs. Rhabdomyosarcomas, inoculated in testes, intensively dessiminated in the lymph-nodes and in the organs of abdomen. It is concluded that the degree of tumor autonomy depends on the tumor--organism interactions.