R. Osieka

Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ABL-negative myeloproliferative disorders

An acquired autoactivating mutation with a V617F amino-acid substitution in the JAK2 tyrosine kinase is frequently found in BCR/ABL-negative myeloproliferative disorders (MPD). Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. In this study, we determined the DNA methylation status of 13 cancer-related genes in the context of JAK2 mutations in 39 patients with MPD. Genes analyzed for hypermethylation were SOCS-1, SHP-1, E-cadherin, MGMT, TIMP-2, TIMP-3, p15, p16, p73, DAPK1, RASSF1A, RARβ2 and hMLH1. We found at least one hypermethylated gene in 15/39 MPD patient specimens, and in 6/39 samples aberrant methylation of the negative cytokine regulator SOCS-1 was present. The JAK2V617F mutation was found in 21/39 patients as determined by allele-specific polymerase chain reaction. Hypermethylation of SOCS-1 was observed in 3/21 patients with an autoactivating JAK2 mutation and in 3/18 patients with wild-type JAK2. Our results suggest that epigenetic inactivation of SOCS-1 may be a complementary mechanism to the JAK2V617F mutation in the pathogenesis of MPD that leads to dysregulation of JAK-STAT signal transduction and thus contributes to growth factor hypersensitivity.

Apoptosis and resistance to daunorubicin in human leukemic cells.

We compared test methods based on specific mechanisms of daunorubicin (DNR) resistance to more global procedures. Assessment of P-glycoprotein (P-gp) expression and function by means of immunocytochemistry, DNR accumulation, and modulation of resistance and accumulation by the P-gp inhibitor cyclosporin A (CsA) were selected as parameters for multidrug resistance (MDR). On the other hand, we used the MTT assay and measured apoptosis and proliferative activity (S- and G2M-phases of the cell cycle) by flow cytometry. Validation of test methods was achieved for four leukemic cell lines (HL-60, KG-1a, K562/WT, K562/ADM). This battery of tests was then applied to mononuclear cells (MNC) from 18 leukemic patients. Low proficiency of MNC to undergo apoptosis and low proliferative activity rather than P-gp-mediated MDR correlated with DNR resistance as measured by the MTT assay. Bell-shaped dose-response curves for apoptosis, however, which reflect a switch from the apoptotic to the necrotic death mode with increasing cellular damage tend to limit practicability in clinical testing, because appropriate dose range and time points need to be explored. Thus, measurement of apoptosis by flow cytometry may be less convenient than the MTT assay for determination of chemosensitivity, if clinical samples with unknown patterns of responsiveness are to be tested. Spontaneous apoptosis in untreated MNC following 24 h incubation in vitro correlated significantly with DNR sensitivity in the MTT assay. A lack of essential viability factors (eg growth factors or cytokines) in vitro which are known to prevent apoptosis may contribute to DNR sensitivity.

Influence of ring substituents on the antitumor effect of dichloro(1,2-diphenylethylenediamine)platinum(II) complexes

SummaryDiastereomeric para-substituted dichloro-(1,2-diphenylethylenediamine)platinum(II) complexes were synthesized and tested for their antitumor activity on the human MDA-MB 231 breast cancer cell line and the P 388 leukemia of the mouse. An interaction with the DNA was demonstrated by UV difference spectroscopy. The D,L configurated, 4-fluoro-substituted complex was the most active.

Expression of apoptosis-related oncoproteins and modulation of apoptosis by caffeine in human leukemic cells

We investigated the modulation of radio- and chemoresistance by caffeine and mechanisms of resistance in human leukemic cell lines and mononuclear cells from 18 leukemic patients. Caffeine synergistically potentiated cytotoxicity and apoptosis induced by ionizing radiation or carboplatin (CPt), but attenuated induction of apoptosis by daunorubicin (DNR) in KG-1a cells. Since caffeine released irradiated as well as DNR-treated KG-1a cells from G2M cell cycle arrest and CPt-treated cells from S-phase arrest, this release does not fully explain the different effects of caffeine. Caffeine synergistically reduced the level of the apoptosis inhibitor glutatione after irradiation or CPt treatment. In contrast, treatment with DNR plus caffeine diminished glutathione levels to a lesser extent than DNR alone. We conclude that the effect of caffeine on glutathione depletion represents a mechanism of action by which caffeine can modulate apoptosis. Caffeine increased CPt cytotoxicity in K562 cells and its doxorubicin-resistant subline (K562/ADM), but little effect was seen in HL-60 cells or mononuclear cells from leukemic patients. Multivariate cluster analysis revealed an association of CPt resistance with the expression of c-Fos, c-N-Ras, and p53 oncoproteins and with proliferative activity (S-phase of cell cycle), but not with Bcl-2 expression.

[dl-1,2-Bis(2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II), a new compound for the therapy of ovarian cancer

SummaryThe synthesis of diastereoisomeric [1,2-bis (2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) complexes,dl-3-PtCl2 andmeso-3-PtCl2, and their evaluation on the hormone-independent, human MDA-MB231 breast cancer cell line, on the cisplatin-sensitive and -resistant L1210 leukemia cell line, on the cisplatinresistant human NIH:OVCAR 3 ovarian cancer cell line, on the P-388 leukemia of the mouse and on the cisplatinsensitive and -resistant Ehrlich ascites tumor of the mouse are described. On all tumor modelsdl-3-PtCl2 produces a marked inhibitory effect. The diastereoisomermeso-3-PtCl2 is less active and more toxic. It is striking thatdl-3-PtCl2 leads to a pronounced inhibition of all cisplatin-resistant tumors. At non-toxic concentrationsdl-3-PtCl2 produces cytocidal effects on the NIH:OV-CAR 3 cell line. Thereforedl-3-PtCl2 is of interest for further evaluation for the therapy of ovarian cancer.

Pseudotumor cerebri after treatment of relapsed acute promyelocytic leukemia with arsenic trioxide.

Pseudotumor cerebri after treatment of relapsed acute promyelocytic leukemia with arsenic trioxide

Sister chromatid exchange-inducing DNA lesions and depression of activation markers on the surface of cultured peripheral blood mononuclear cells after the addition of streptococcal pyrogenic exotoxins A and C.

Cultivation of peripheral blood mononuclear cells (PBMC) in the presence of streptococcal pyrogenic exotoxins (SPE) A and C resulted in a significant induction of sister chromatid exchange (SCE)-inducing DNA lesions. Concomitantly, the expression of interleukin-2 receptor α chain (IL-2Rα chain), transferrin receptor (TfR), and major histocompatibility complex class II molecule HLA-DR on the surface of phytohemagglutinin-activated T cells from whole blood culture cells (WBCC) significantly decreased within 72 h, that is at least two cell cycles, whereas unstimulated T cells from WBCC did not express these markers but had lost their CD3 molecules, an effect reported to precede apoptosis as part of a T cell inactivation pathway. However, no apoptotic cells were observed within a cultivation period of 120 h. We observed clearcut differences in the responses towards SPE A in WBCC and isolated lymphocytes, since SPE A-treated lymphocytes showed an increase in the [3H]-thymidine incorporation and did express IL-2Rαchain and TfR on their cell surface. Regardless of the precise underlying mechanism, T cells from WBCC seem to be in a state of functional incompetence. The data presented here are the first to provide strong evidence that streptococcal toxins produce SCE-inducing DNA lesions in PBMC, an effect that might contribute to the process of immune cell lethality in streptococcal toxic shock-like syndrome and could be of pivotal importance in the pathogenesis of severe streptococcal disease.