Martin Tiefenthaler

Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides.

Currently available methods for treatment of human prostatic carcinoma aim to inactivate the androgen receptor (AR) by androgen deprivation or blockade with anti-androgens. Failure of endocrine therapy and tumor progression is characterized by androgen-independent growth despite high levels of AR expression in metastatic disease. We inhibited AR expression in LNCaP prostate tumor cells by using antisense AR oligodeoxynucleotides (ODNs) and explored whether antisense AR treatment would be conceivable as a therapy for advanced prostate cancer. Among the various AR antisense ODNs tested, a 15-base ODN targeting the CAG repeats encoding the poly-glutamine region of the AR (as750/15) was found to be most effective. Treatment of LNCaP cells with as750/15 reduced AR expression to ∼2% within 24 hours compared with mock-treated controls. AR down-regulation resulted in significant cell growth inhibition, strongly reduced secretion of the androgen-regulated prostate-specific antigen, reduction of epidermal growth factor receptor expression, and an increase in apoptotic cells. Mis-sense and mismatched control ODNs had no or only slight effects. Antisense inhibition was also very efficient in LNCaP-abl cells, a subline established after long-term androgen ablation of LNCaP cells, resulting in inhibition of AR expression and cell proliferation that was similar to that seen for parental LNCaP cells. This study shows that inhibition of AR expression by antisense AR ODNs may be a promising new approach for treatment of advanced human prostate cancer.

AGE DEPENDENT APOPTOSIS AND LOSS OF RHABDOSPHINCTER CELLS

PURPOSE To our knowledge the exact age dependent morphological and functional changes of the sphincter mechanism have not been investigated. Therefore, cell densities of the urethra and the urethral rhabdosphincter across various age groups, and the appearance of apoptosis were examined to explore the changes in these structures during the aging process. MATERIALS AND METHODS Specimens were obtained from 16 male and 7 female cadavers 5 weeks to 92 years old. Histological sections were taken from 3 different levels of the rhabdosphincter and urethra. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method was used to detect apoptosis in the urethra and rhabdosphincter. In all specimens relative volume densities of the striated muscle fibers, apoptotic indexes and diameters of the rhabdosphincter and urethra were determined. RESULTS An age dependent increase of apoptosis of the striated muscle fibers of the rhabdosphincter led to a dramatic decrease in the number of striated muscle cells. In the 5-week-old neonate 87.6% and in the 91-year-old woman 34.2% of the rhabdosphincter consisted of striated muscle cells. Overall, a direct linear correlation between the age of the specimens and decrease in volume densities of the striated muscle cells was evident. CONCLUSIONS The dramatic decrease in the number of striated muscle cells in the rhabdosphincter of the elderly due to apoptosis represents the morphological basis for the high incidence of stress incontinence in this population.

Urinary incontinence in the elderly and age-dependent apoptosis of rhabdosphincter cells

With advancing age, a progressive and age-dependent decrease of the density of striated muscle cells can be observed in the rhabdosphincter. This continuous loss of striated muscle cells due to apoptosis may finally lead to urinary incontinence.

The immunomodulator FTY720 interferes with effector functions of human monocyte-derived dendritic cells

The potent immunomodulator FTY720 elicits immunosuppression via acting on sphingosine 1‐phosphate receptors (S1PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte‐derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720‐P) and investigated their influence on DC surface marker expression, protein levels of S1PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th‐priming capacity. We report that both FTY720 and FTY720‐P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720‐P showed an impaired immunostimmulatory capacity and reduced IL‐12 but increased IL‐10 production. T cells cultured in the presence of FTY720‐ or FTY720‐P‐treated DC showed an altered cytokine production profile indicating a shift from Th1 toward Th2 differentiation. In treated immature and mature DC, expression levels for two S1PR proteins, S1P1 and S1P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen‐presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid‐derived drugs.

Sphingosine-1-phosphate receptor type-1 agonism impairs blood dendritic cell chemotaxis and skin dendritic cell migration to lymph nodes under inflammatory conditions

SEW2871 is a potent sphingosine-1-phosphate receptor type-1 (S1P(1))-selective agonist that induces peripheral lymphopenia through sequestration of lymphocytes into secondary lymphoid organs, similar to the non-selective sphingosine-1-phosphate (S1P) receptor agonist FTY720. FTY720 has been reported to interfere with human dendritic cell (DC) effector functions and both FTY720 and SEW2871 have been shown to modulate murine DC trafficking in vivo. Little is known about the possible effects of SEW2871 on human and murine DC functions. Here, we demonstrate that in contrast to FTY720, SEW2871 does not induce down-regulation of S1P(1) in human DCs and thus does not exert a functional antagonism at S1P(1). Notably, the compound was found to impair chemotaxis of immature and mature human DCs in vitro, possibly by interfering with the activation of p44/p42 and p38 mitogen-activated protein kinase signaling pathways. Comparative FACS analyses show that SEW2871 mediates CD18 down-regulation on mature human DCs. The influence on DC migration could be confirmed with in vivo assays using BALB/c mice in which SEW2871 impairs the migration of CD11c+ DC and CD207+ Langerhans cells (LC) to the draining lymph nodes (LNs) under inflammatory conditions. These results suggest that the S1P-S1P(1) axis might not only control lymphocyte trafficking but also play a pivotal role in DC migration from the skin to LN.

Increased lactate production follows loss of mitochondrial membrane potential during apoptosis of human leukaemia cells

Acute tumour‐lysis syndrome (ATLS) is a frequently fatal complication after cytoreductive leukaemia therapy. Lactic acidosis is associated with ATLS and its extent is correlated with the severity of ATLS. In the course of cytoreductive therapy, apoptosis is induced in tumour cells, which results in loss of mitochondrial function. We hypothesize that loss of mitochondrial function leads to compensatory glycolysis, which is the main cause of lactate accumulation and acidosis. We tested this hypothesis using the model of glucocorticoid‐induced apoptosis in the human acute lymphoblastic leukaemia cell line CCRF‐CEM. After induction of glucocorticoid‐induced apoptosis, a biphasic course of lactate production was observed. Prior to the onset of apoptosis, i.e. prior to the loss of membrane potential, lactate production was reduced. However, subsequent to loss of mitochondrial membrane potential a massive increase in lactate production was observed (15·5 ± 0·5 versus 10·17 ± 0·09 mmol/106 cells, P = 0·001). We also demonstrated that inhibition of respiratory chain activity by antimycin A resulted in excess lactate production. In the model cell line used, conditional bcl‐2 expression delayed glucocorticoid‐induced apoptosis by protecting against loss of mitochondrial membrane potential; bcl‐2 expression delayed the increase in lactate production and had no effect on the pre‐apoptotic drop in lactate production. Apoptosis‐induced lactate production was also observed in other cell lines (HL60, THP1 and OPM2) with various cytotoxic agents [doxorubicin, gemcitabine and vumon (VM26)]. Thus, the data suggest that lactate acidosis can be caused by apoptotic loss of mitochondrial function and massive apoptosis of a tumour mass via lactic acidosis may be the essential pathological event in ATLS.

Differential effects of cladribine and gemcitabine on erythroid and granulocytic progenitors from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal neoplastic disease that originates in a pluripotent stem cell. Selection of normal progenitors by graft-purging may improve the outcome after autologous transplantation. In our methylcellulose assays, the nucleoside analogs cladribine (2-CdA) and gemcitabine (dFdC) showed more prominent inhibitory effects on CML than normal bone marrow (BM) progenitors. For dFdC, however, long-term incubations were necessary to achieve complete inhibition. Deoxycytidine kinase, the key enzyme of both 2-CdA and dFdC metabolisms, was only partially responsible for this differential sensitivity. We suggest that 2-CdA and dFdC might be helpful in purging of CML BM cells before autologous BM transplantation. Further studies on more primitive cells are warranted.

Delayed Addition of Deoxycytidine Protects Normal CD34+ Cells against Cytotoxicity of Gemcitabine without Compromising Its Activity against Human Leukemic Cells

In phase I and II clinical trials, the deoxycytidine analogue 2′,2′ difluorodeoxycytidine (dFdC, gemcitabine) has shown promising antitumor activity in leukemia as well as in solid tumors. Preclinical and clinical studies of gemcitabine suggested that myelosuppression was the dose‐limiting toxicity. The present investigations were designed to test the effect of continuously administered gemcitabine on the in vitro clonal growth of normal CD34+ cells isolated from peripheral blood and the promyelocytic cell line, HL‐60. For this purpose, CD34+ and HL‐60 cells were cultured in methylcellulose in the continuous presence of 0.1–16 nM of gemcitabine. The results show a dose‐dependent inhibition of colony growth of normal as well as leukemic cells. However, HL‐60 cells were up to 12‐fold more sensitive towards gemcitabine than normal progenitors. For rescue experiments, the natural pyrimidine deoxycytidine (dCyd) was added to CD34+ and HL‐60 cells simultaneously or with delay. Coadministration of 1mM dCyd to separate cultures resulted in complete restoration of colony formation capacity of CD34+ and HL‐60 cells. Delayed addition of 1 mM dCyd after 48 and 72 hours recovered up to 90% and 40%, respectively, of stem cell proliferation, whereas HL‐60 cells remained substantially inhibited (4.5% ± 3.5% versus 0%). Delayed addition after 48 and 72 hours protected about 80% and 50%, respectively, of myelopoietic and erythropoetic colony formation, whereas colony formation obtained from HL‐60 cells remained significantly inhibited (9.6% ± 4.17% versus 0%). These in vitro data suggest that there is a marked difference in the susceptibility of leukemic and normal CD34+ cells to gemcitabine and that delayed administration of dCyd may further reduce the bone marrow cytotoxicity of gemcitabine without impairing its antitumor effect.

APOPTOSIS OF RHABDOSPHINCTER CELLS: THE MAIN CAUSE OF URINARY INCONTINENCE WITH ADVANCING AGE?

Reproduction Form B-1 InStitUtii I UNIVERSITY OF INNSBRUCK, INNSBRUCK, AUSTRIA I