Growth hormone interferes with the progression of myocarditis in rats.

In this study, we investigated whether recombinant human growth hormone (rhGH) influences the progression of myocarditis. We induced experimental autoimmune myocarditis in F344 rats by subcutaneous injection of cardiac myosin, and divided the rats into three groups: (1) control group, saline injection; (2) pre-treated group, subcutaneous injection of rhGH (100 mIU/rat/day for 10 days) before induction of experimental autoimmune myocarditis; and (3) post-treated group, subcutaneous injection of rhGH (100 mIU/rat/day for 10 days) after induction of experimental autoimmune myocarditis. On the 35th day after induction of experimental autoimmune myocarditis, all rats were sacrificed and the hearts were examined. The increase in body weight was smaller in the control group than the pre-treated group and the rate of heart weight/body weight was larger in the control group than in the two treated groups. Histopathologically, rats in the control group showed multifocal infiltration by inflammatory cells, mainly neutrophils, lymphocytes and macrophages, extensive fibrosis, and a higher proportion of mast cells in the inflamed region. In contrast, rats in the two treated groups showed only minor changes. We found that rhGH did not influence the distribution of lymphocytes in peripheral blood in the three groups, and that rhGH induced G1 checkpoint dysfunction, thereby arresting the cell cycle in G1 and inhibiting the proliferation of mast cells in vitro. These findings suggest a possible role for mast cells in the progression of myocarditis and the rhGH may be a candidate for use as a new tool to treat myocarditis.

Methimazole interferes with the progression of experimental autoimmune myocarditis in rats.

In order to ascertain whether methimazole, a drug commonly used for the treatment of hyperthyroidism, interferes with the progression of autoimmune-mediated myocardial injury, we investigated the effect of methimazole on experimental autoimmune myocarditis (EAM) in rats. EAM was induced by immunization with porcine cardiac myosin. Methimazole administration markedly slowed the body weight growth in both normal and EAM rats, but did not induce morphologic change of cardiac tissue in normal rats. In EAM rats, macroscopic examination revealed discoloration of the cardiac surface, and histopathological examination by light microscopy showed extensive myocardial necrosis, infiltration by inflammatory cells and myocardial fibrosis. In the EAM rats treated with methimazole, the discolored areas on the cardiac surface were markedly diminished in size, and the myocardial necrosis, cellular infiltration and fibrosis were significantly less severe. To identify the mechanism responsible of this effect, we investigated the change of regulatory lymphocyte subsets in peripheral blood using an immunofluorescence technique with a flow cytometer. A decrease in the helper/suppressor T cell ratio as a result of the increased proportion of suppressor T cells and a decrease in the proportion of B cells were observed in normal rats after methimazole administration, and similar findings were made in the EAM rats treated with methimazole. These results indicate that methimazole interferes with the progression of EAM, and immunosuppression may, at least in part, be involved in the inhibitory effect of methimazole on EAM in rats.

Different responses of polyploidized V79 cells after removal of two drugs, demecolcine and K-252a.

To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by demecolcine and K-252a, inhibitors of spindle-fiber formation and protein kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a week. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line.

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression.

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine.

The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.

Apoptotic cell death of high polyploid cells in a cultured sarcoma cell line.

It is well known that DNA-ploidy is useful independent prognosticator of malignancy. However, the biological significance of polyploid cells and the relation between polyploidy and prognosis is not well understood. We analyzed DNA ploidy by flow cytometry in Meth-A cells (a cultured sarcoma cell line) after treatment with K252a, a protein kinase inhibitor, and showed induction of polyploidization. Apoptotic cell death of the high polyploid cells was verified by flow cytometry, morphological observation and gel analysis of DNA integrity. Expression of tumor-suppressor nuclear protein p53 investigated by immunohistochemistry was increased 10-fold or more in cells with 16C (C = haploid DNA content) relative to cells with 2C, suggesting that the overexpression of p53 was involved in the apoptosis. These results may be of clinical relevance since it has been known that both DNA ploidy and p53 expression have prognostic significance.

Spontaneous polyploidization results in apoptosis in a Meth-A tumor cell line.

Cultured Meth-A cells always include a small fraction of large cells, which had a DNA content above 4c (polyploid cells). The process from the formation to the disintegration of polyploid Meth-A cells was measured by means of time-lapse videography. Polyploid Meth-A cells arose spontaneously from normal cells (polyploidization), then died by apoptosis. The fraction of polyploid cells gradually increased in seven day-exponential cultures with a low concentration of demecolcine, which is a specific inhibitor of cell division. The results revealed that the polyploid Meth-A cells are generated from normal cells by failing cell division and that they die by apoptosis.

Characterization of T cell receptor beta chains of accumulating T cells in skin allografts in mice.

The study of T cells involved in the immune reaction that occurs in engrafted organs should provide information that would be helpful in the regulation of allograft rejection in organ transplantation. Toward this end, we focused on detection and characterization of accumulating T cells in mouse skin allografts from B10.A(4R) to C57BL/6 mice in vivo. T cell receptor beta genes were amplified by reverse transcriptase-PCR from mRNA of the skin grafts, and accumulating T cell receptor beta gene clonotypes were identified by their single strand conformation polymorphism. Their joining region usage and the amino acid sequences of the complementarity-determining region-3 were then determined. The results were as follows: (1) Distinct oligoclonal accumulation of T cells was more prevalent in the skin allografts than in the syngenic skin grafts. (2) Although the accumulating T cell clonotypes appeared to use many different variable-region gene families, preferential combinations of variable region-joining region were found. (3) Several homologous amino acid sequences were found in these accumulating TCR beta genes in allografts, suggesting that these T cells are driven by the same or similar antigens. (4) In addition, little T cell accumulation was found in spleens from the mice with allografts or syngenic skin grafts. Taken together, accumulating T cells in the skin allografts were detected in vivo, and some appeared to have characteristics in common. This may lead to T cell clonotype-specific therapy in organ transplantation.

The important role of PKC in controlling polyploidy formation in cultured fibrosarcoma cell line.

Induction of polyploidization by colcemid in cultured fibrosarcoma cells (Meth-A cells) was examined. Activators of protein kinase C (PKC), phorbol 12-myristate 13-acetate (PMA) and ATP, inhibited colcemid-induced polyploidization, but not colcemid-induced cell proliferation cessation. These findings suggest that a down-regulation of PKC activity results in checkpoint "dysfunction" which induces polyploidization and that inhibition of polyploidization induction by PMA and ATP is not a result of the inhibition of colcemid-induced depolymerization of tubulin.

Apoptosis by demecolcine in V79 cells.

Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01 microgram/ml demecolcine, V79 cells proliferated exponentially as well as controls, although temporal M phase accumulation occurred 6 h after the addition of demecolcine. At 0.1 microgram/ml, the cells became hyperploid after remaining in the M phase for some time. Apoptosis occurred in V79 cells exposed to demecolcine at a concentration of 0.03 microgram/ml. Apoptosis was defined as the appearance of a sub-G1 peak in DNA histograms and a ladder pattern of fragmented DNA in gelelectrophoresis.

Potentiation of K252a, a protein kinase inhibitor-induced polyploidization by cAMP in cultured fibrosarcoma cell line.

We found that K252a, a potent inhibitor of protein kinases (PK), induced DNA re-replication of Meth-A cells, i.e., DNA synthesis at a higher DNA ploidy without undergoing cytokinesis (polyploidization). The K252a-induced polyploidization was inhibited by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, suggesting that the polyploidization is caused through inhibition of PKC. By contrast, the polyploidization was potentiated by adenosine 3':5'-cyclic monophosphate (cAMP), a cAMP-dependent protein kinase (PKA) activator. These findings suggest that the cAMP-dependent signaling pathway and diacylglycerol (DAG)-dependent signaling pathway play an important role in regulating the induction of polyploidization in Meth-A cells, through a possible "cross-talk" between the two pathways.

Plant-glycoside modulation of cell surface related to control of differentiation in cultured B16 melanoma cells.

We have shown that the ginsenosides Rh1 and Rh2, which are plant glycosides with a dammarane skeleton resembling a steroid skeleton as an aglycone, control the phenotypic expression of mouse B16 melanoma cells in different ways. The effects of Rh1 and Rh2 on the cell surface were studied to clarify the relationship between the control of phenotypic expression and modification of the cell surface in B16 melanoma cells. Rh2, which has the capacity to inhibit the growth of and to stimulate melanogenesis in B16 melanoma cells, causes flattening of the cells cultured in a collagen gel, leading to organized, nonoverlapping monolayers. Cell-to-cell adhesiveness and cell-to-substrate adhesiveness were markedly increased in the B16 melanoma cells treated with Rh2. In Rh2-treated cells, the binding of peanut agglutinin on the cell surface was also increased, whereas no marked changes were observed in the binding of concanavalin A or wheat germ agglutinin. In contrast, Rh1, which showed no effect on cell growth, but did stimulate melanogenesis, did not cause morphological changes of the cells and exerted no effect on cell adhesiveness or cell surface lectin binding. 1,6-Diphenyl-1,3,5-hexatriene polarization values markedly decreased in cells treated with either Rh1 or Rh2. Rh2 was found to be incorporated in the lipid fraction of the B16 melanoma cell membrane. In contrast, Rh1 was not detected in the lipid fraction of B16 melanoma cells. However, novel lipid components were found.