In vitro induction of apoptosis of neoplastic cells in low-grade non-Hodgkin's lymphomas using combinations of established cytotoxic drugs with bendamustine.

BACKGROUND AND OBJECTIVES: Regulation of apoptotic cell death is being increasingly recognized as a mechanisms by which cytostatic agents mediate tumor cell death. Preliminary clinical studies with bendamustine, an alkylating agent with a purine nucleus, provide strong evidence that this drug is a highly effective cytostatic in low grade lymphomas. Therefore, we investigated the in vitro activity of bendamustine in combination with other established cytotoxic drugs. DESIGN AND METHODS: 2 lines (DOHH-2, WSU-NHL) and mononuclear cells (MNC) from patients with leukemic low-grade B-non-Hodgkin's lymphoma (NHL) (n=10), T-NHL (n=7) and chronic lymphocytic leukemia (CLL) (n=12). Apoptosis (7-AAD), depolarization of mitochondrial membrane potential (MMP, JC-1), caspase-3-activity (FIENA) and cell proliferation (XTT/WST-1) were determined. Several incubation times and drug dosages (for IC(30/50/75/90)) were studied. Synergistic, additive or antagonistic effects were calculated by a median plot effect and the combination index (CI) method. RESULTS: In general, combinations of bendamustine with mitoxantrone or doxorubicin resulted in antagonistic effects in the tested cell lines and the MNC from the patients. CI-calculation failed in these cases since there was not a sufficient dose response. On the other hand, the combination of bendamustine with 2-CdA showed synergistic in vitro activity on the tested cell lines, neoplastic lymphocytes from patients with peripheral T-cell lymphomas and partially on MNC from patients with CLL and B-NHL. The antagonism of the combination of bendamustine and anthracyclines appeared to be due to inhibition of depolarization of mitochondrial-membrane potential and caspase-3-activity during apoptosis of the studied cell lines. INTERPRETATION AND CONCLUSIONS: In conclusion, our results suggest that schedules using combinations of bendamustine and anthracyclines should not be recommended for the treatment of low-grade NHL, whereas bendamustine combined with 2-CdA could be considered for the development of future treatment strategies.

Deregulated expression of prostate apoptosis response gene-4 in less differentiated lymphocytes and inverse expressional patterns of par-4 and bcl-2 in acute lymphocytic leukemia.

INTRODUCTION: Prostate apoptosis response gene-4, known as par-4, is a new proapoptotic factor functionally required but not sufficient for apoptosis. Since there is evidence from prostate cancer cells that par-4 is involved in regulation of bcl-2 we assessed expression of par-4 and bcl-2 in different populations of normal and neoplastic lymphocytes. MATERIALS AND METHODS: Expression of par-4 mRNA and protein in different subpopulations of normal and neoplastic lymphocytes was assessed by reverse transcription polymerase chain reaction and Western blot. RESULTS: Par-4 mRNA was not detectable in lymphocytes of healthy volunteers (n = 10), but was present in the majority of samples of chronic lymphocytic leukemia (n = 30), chronic lymphocytic leukemia/prolymphocytic leukemia (n = 6) and acute lymphocytic leukemia (n = 10). Par-4 protein was expressed unanimously in samples of mononuclear cells from healthy volunteers and patients with CLL, but less frequently in immature lymphocytes, including neoplastic cells of CLL/PLL and ALL. The decreased frequency of par-4 expression in immature subpopulations was confirmed by results on lymphocytic cell lines at various stages of maturation. Comparing the expressional patterns of par-4 and bcl-2 there was an inverse relationship of both proteins in ALL and different lymphocytic cell lines, indicating a functional relationship of par-4 and bcl-2. CONCLUSIONS: This study establishes par-4 as a factor expressed in the majority of normal and neoplastic lymphocytic cells, demonstrating a decreased frequency of protein expression in less differentiated lymphocytes and an inverse expressional pattern of par-4 and bcl-2 in lymphocytic cell lines and ALL.