C. D. Gerharz

Tumor dedifferentiation: An important step in tumor invasion

Tumor invasionin vivo was studied by light and electron microscopy as well as by immunofluorescence microscopy. Special regard was paid to the grade of tumor differentiation. Dimethylhydrazine-induced murine colonic carcinomas comprising a differentiated and an undifferentiated tumor type with low and high invasiveness respectively, were used. At the invasion front of both tumor types a striking dissociation of the organized tumor cell complexes into isolated tumor cells was found together with a loss of most of the cytological features of differentiation. It is supposed that this process mobilizes the tumor cells from the main tumor bulk enabling them to invade the host tissue by active locomotion. This view is strongly supported by the demonstration of morphological equivalents of active cell movement such as pseudopodia-like cytoplasmic extrusions, adaptive changes of the cell shape and microfilament bundles. Although the proposed mechanism of tumor invasion is essentially the same in both tumor types, the grade of differentiation is nevertheless critical, as in the undifferentiated carcinomas only subtle dedifferentiation steps (loss of basement membrane and cell junctions) are necessary to acquire an invasive status. This fact may explain the comparatively high invasiveness and poor prognosis of undifferentiated carcinomas.

Accumulation of hydroxyethyl starch (HES) in the liver of patients with renal failure and portal hypertension

Hydroxyethyl starch (HES) has gained wide clinical acceptance as a colloidal plasma substitute. We were able to study the liver biopsies of two patients with renal failure who developed ascites after repeated infusions of HES. All types of liver cells displayed massive accumulation of HES with the morphologic resemblance to a storage disease. These changes could be distinguished clearly from the lesions of a hereditary disorder by light and electron microscopy. Although it is difficult to establish a causative role for HES in the development of ascites on the bases of morphological changes alone, one should be cautious about giving HES to patients with renal failure until exact data from further experimental studies are available.

Secretion of GM-CSF and M-CSF by human renal cell carcinomas of different histologic types

OBJECTIVES To analyze the secretion of hematopoietic growth factors and the expression of their corresponding receptors in 40 newly established renal cell carcinoma (RCC) cell lines of different histologic types. Little is known about the secretion and function of hematopoietic growth factors by human RCCs. METHODS The expression of the hematopoietic growth factors (ie, erythropoietin, interleukin [IL]-3, IL-5, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], and macrophage colony-stimulating factor [M-CSF]) was determined by enzyme-linked immunosorbent assay analysis under different culture conditions, including suspension culture and monolayer cultures (plastic and Matrigel-coated culture flasks). The expression of their corresponding receptors was defined by fluorescence activated cell scanner analysis and by reverse-transcriptase polymerase chain reaction. The response of the RCC cell lines to exogenous hematopoietic growth factors was analyzed by MTT assay. RESULTS In almost all of the cell lines, significant amounts of GM-CSF and M-CSF were secreted, and in four cell lines, a secretion of G-CSF was detected. Fourteen RCC cell lines showed secretion of IL-3, and production of IL-5 and erythropoietin was not observed in any cell line. Secretion of GM-CSF and M-CSF was affected by the substratum offered for cell attachment in the adherent cultures. GM-CSF secretion was more pronounced under culture conditions with a reduced frequency of cell-to-cell contacts. Two cell lines were shown to express receptors for M-CSF, but receptors for G-CSF and GM-CSF could not be detected in any cell line. Exposure to exogenous G-CSF, GM-CSF, and M-CSF did not affect the proliferation of our RCC cell lines. CONCLUSIONS The results of our study clearly demonstrate that human RCC cells can secrete significant amounts of G-CSF, GM-CSF, M-CSF, and IL-3, and are thereby theoretically able to modulate the hosts tumor-directed immune response.

The intraclonal and interclonal phenotypic heterogeneity in a rhabdomyosarcoma cell line with abortive imitation of embryonic myogenesis

SummaryThree distinct subpopulations (A, B, C) derived from a dimethylbenzanthracene-induced rat rhabdomyosarcoma were established as permanent cell lines. Although the clonal nature of each of these subpopulations was confirmed by repeated recloning procedures, a striking intraclonal phenotypic heterogeneity was observed. By means of immunofluorescence microscopy and transmission electron microscopy, it could be shown that these subpopulations closely recapitulate stages of embryonic rhabdomyogenesis both in vitro and in vivo, but differ in their particular range of maximum differentiation. Embryonic rhabdomyogenesis is imitated most perfectly by subpopulation C, in which multinuclear myotubes are formed in vitro by fusion of mononuclear cells, and α-sarcomeric actin is expressed in the multinuclear cells and in a few mononuclear cells. After retransplantation in vivo, subpopulation C further proceeds in fine structural differentiation, now exhibiting myofibrils with a sarcomeric organization in the myotube-like giant cells. The cells of subpopulation B do not exceed the stage of mononuclear desmin-positive cells in vitro, but synthesize thin and thick myofilaments after retransplantation in vivo. The cells of subpopulation A recapitulate embryonic rhabdomyogenesis least successfully being confined to the stage of mononuclear desmin-positive cells. Thus, the coexistence of diverse subpopulations and the cellular maturation within these subpopulations together contribute to the phenotypic heterogeneity of rhabdomyosarcomas.

Fetal calf serum and retinoic acid affect proliferation and terminal differentiation of a rat rhabdomyosarcoma cell line (BA-HAN-1C).

We report on the establishment of a model for differentiation induction in sarcomas, using the clonal rhabdomyosarcoma cell line BA-HAN-1C. This rhabdomyosarcoma cell line is composed of morphologically undifferentiated mononuclear stem cells, some of which spontaneously fuse to form terminally differentiated multinuclear myotube-like giant cells. The deprivation of fetal calf serum (FCS) or the exposure to retinoic acid, respectively, resulted in a significant inhibition of proliferation (P less than 0.001) and a marked increase in cellular differentiation as shown by a significant increase in the number of myotube-like giant cells (P less than 0.001) and in the creatine kinase activity (P less than 0.05) used as a biochemical marker of differentiation. Furthermore, after exposure to retinoic acid about 30% of the mononuclear tumour cells exhibited morphological features of rhabdomyogenic differentiation, such as bundles of thick and thin myofilaments, which had never been observed in the mononuclear cells of untreated cultures. These results confirm that the inverse linkage between proliferation and differentiation known from embryonic myogenesis is preserved in our rhabdomyosarcoma cell line. The failure to induce terminal differentiation by exposure to retinoic acid in all the cells of our clonal cell line indicates that some tumour cells might epigenetically be blocked from responding to retinoic acid. The temporary growth retardation observed after FCS-deprivation suggests that autocrine stimulation of proliferation may be operating in our cell line, too.

Terminally differentiated postmitotic tumor cells in a rat rhabdomyosarcoma cell line

SummaryA permanent rat rhabdomyosarcoma cell line (BA-HAN-1C) has been established, the phenotype of which is characterized by the coexistence of undifferentiated mononuclear cells and differentiated multinuclear myotube-like giant cells. The failure of attempts to separate these two cell types by repeated recloning procedures indicates their close histogenetic relationship and suggests that differentiation in this tumor proceeds in a similar manner to that in normal striated muscle where postmitotic myotubes arise from mononuclear myoblasts by fusion. The morphologically undifferentiated mononuclear tumor cells were shown to be actively proliferating and to incorporate thymidine methyl-3H(3H-TdR). The myotubelike giant cells neither incorporated3H-TdR nor underwent mitosis or exhibited any clonogenic potential. After retransplantation into syngenic rats, tumor growth was markedly retarded when the tumor cell inoculum contained a high percentage of myotube-like giant cells. These data show that proliferative activity in this rhabdomyosarcoma cell line is confined to the mononuclear tumor cell compartment, the multinuclear myotube-like giant cells having withdrawn from the cell cycle and represent terminally differentiated postmitotic cells. This cell line should provide a valuable tool for further investigation of coherent aspects of proliferation and differentiation using various differentiation inducers.

The nature of host tissue destruction in tumor invasion. An experimental investigation on carcinoma and sarcoma xenotransplants.

SummaryThe nature of host tissue destruction in tumor invasion was investigated in experimentally induced carcinomas and sarcomas, xenografted into skeletal muscle. By means of light and electron microscopy it was shown that in both carcinomas and sarcomas the confrontation of host tissue with the invading tumor cells does not result in immediate destruction of host tissue but in a transitory state of coexistence which gradually proceeds to progressive host tissue atrophy. This process of progressive atrophy, which finally results in the total disappearance of the invaded host tissue, is considered to be caused mainly by the increasing pressure and competitive withdrawal of oxygen and nutrients by the invading and proliferating tumor cells. Morphological changes suggesting an active enzymatic breakdown of host tissue cells by tumor cells were not observed during any stage of tumor invasion.

Heterogeneous response to differentiation induction with different polar compounds in a clonal rat rhabdomyosarcoma cell line (BA-HAN-1C).

The clonal rat rhabdomyosarcoma cell line BA-HAN-1C was tested for its susceptibility to differentiation induction with different polar compounds. This cell line is composed of proliferating mononuclear tumour cells, some of which spontaneously fuse to form terminally differentiated postmitotic myotube-like giant cells. Exposure of BA-HAN-1C cells to dimethylsulphoxide (DMSO), hexamethylene bisacetamide (HMBA), sodium butyrate (NaBut) and N-monomethylformamide (NMF) resulted in a significant inhibition of proliferation (P less than 0.001) and in a simultaneous increase in differentiation. The response was most pronounced after exposure to NMF as evidenced by a marked increase in the creatine kinase activity used as a biochemical marker of differentiation (P less than 0.05) and the number of terminally differentiated myotube-like giant cells (P less than 0.001). Furthermore, about 5% of the mononuclear cells exhibited thick and thin myofilaments which were never observed in the mononuclear cells of the control. In contrast, the effects of DMSO, HMBA and NaBut were exclusively confined to a significant increase in biochemical differentiation (P less than 0.05), whereas no increase in morphological differentiation was observed and the number of myotube-like giant cells even significantly (P less than 0.001) decreased. This heterogeneous response to differentiation induction with different polar compounds probably indicates different mechanisms of action and suggests that the induction of biochemical differentiation might be independently regulated from events leading to cell fusion and terminal differentiation.

Uniform response of c-raf expression to differentiation induction and inhibition of proliferation in a rat rhabdomyosarcoma cell line

SummaryThe clonal rat rhabdomyosarcoma cell line BA-HAN-1C is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. Both the induction of differentiation by retinoic acid (RA) and by sodium butyrate (NaBut), as well as the inhibition of proliferation by fetal calf serum (FCS)-depleted medium uniformly resulted in the same effects. There was a significant (p< 0.001) inhibition of proliferation and induction of cellular differentiation, as evidenced by a significant (p<0.05) increase in creatine kinase activity. Furthermore, after exposure to RA-supplemented or FCS-depleted medium, a significant (p< 0.001) increase in the number of myotube-like giant cells was observed. These effects were preceded by a uniform enhancement of c-raf mRNA expression, which became evident 6 h after exposure to RA, NaBut and FCS-depleted media. C-raf mRNA expression persisted at an elevated level throughout the observation period of 5 days after exposure to RA or NaBut, whereas the increased expression of c-raf mRNA observed after FCS-depletion declined near to the basal level after only 24 h. Furthermore, a transient c-fos mRNA expression was observed 15 and 30 min after exposure to RA-supplemented and FCS-depleted medium but not after exposure to NaBut. The present results suggest a possible role of c-raf in the regulation of differentiation and proliferation of this cell line. Since all our experiments with RA, NaBut and FCS-depletion resulted in an early peak of c-raf mRNA expression, it is suggested that this early peak may be sufficient to trigger events crucial for differentiation and proliferation of BA-HAN-1C tumor cells.

The establishment of two rat colonic carcinomas in tissue culture. A basic prerequisite for standardized experiments on the biology of colonic carcinomas in vivo.

SummaryTwo rat colonie carcinomas (DMH-Co-1 and DMH-Co-2) derived from dimethyl-hydrazine-induced metastasizing adenocarcinomas were established as permanent cell lines. By means of electron microscopy, immunofluorescence microscopy and biochemical analysis of cytoskeletal components, it has been shown that both tumor cell lines retain in vitro the phenotypic characteristics of the primary tumors. The in vitro growth properties revealed only minor differences between the two cell lines. After retransplantation in vivo, DMH-Co-2 gave rise to moderately differentiated adenocarcinomas, whereas the tumors arising from DMH-Co-1 exhibited a continuum of differentiation encompassing adenocarcinomas, undifferentiated carcinomas and squamous cell carcinomas. These permanent cell lines offer the opportunity for isolating divergent subpopulations by in vitro cloning and facilitate standardized experiments on their biological behaviour in vivo.