Differential responses of oligodendrocytes to tumor necrosis factor and other pro-apoptotic agents: role of ceramide in apoptosis

Staurosporine induced apoptosis in a human oligodendroglioma cell line (HOG), neonatal rat oligodendrocyte (O2A(+)) precursors, and mature rat oligodendrocytes. In all three cell culture systems, the activation of caspase-3-like activity (CPP32) coincided with the increased formation of ceramide from sphingomyelin and the onset of DNA fragmentation. Further, the addition of exogenous C(2)-ceramide induced CPP32 activation and DNA fragmentation in all three culture systems. Raising endogenous ceramide levels by the addition of the ceramidase inhibitor, oleoylethanolamine, enhanced apoptosis in both a time- and concentration-dependent manner. Inhibitors of phosphatidylinositol 3-kinase (wortmannin and LY294002) also induced caspase-3 (CPP32) activation, increased ceramide formation, induced DNA fragmentation, and reduced cell viability. In contrast, cytokines such as tumor necrosis factor-alpha (TNF-alpha) had a differential effect on the three cell cultures. Thus, TNF-alpha (160 ng/ml) induced 70% apoptosis in 24 hr in freshly isolated rat brain O2A(+) precursor cells, 60% apoptosis in 24 hr in a human oligodendroglioma (HOG) cell line, but no apoptosis in mature neonatal rat oligodendrocytes. Interferon-gamma augmented the activation of CPP32 by TNF-alpha in HOG cells and O2A(+) oligodendrocyte precursor cells but had no effect on mature oligodendrocytes. Thus, the death pathway appears to be similar in the three cell lines but the lack of coupling between TNF-alpha receptors and the apoptotic pathway leads to a lack of response to cytokines in mature oligodendrocytes.     

Synthetic retinoid CD437 induces S-phase arrest and apoptosis in human prostate cancer cells LNCaP and PC-3

BACKGROUND: Exposure of prostate carcinoma cell lines to retinoids, which function through the classical retinoic acid nuclear receptor, (RARs) or retinoid X receptors (RXRs), results in minimal cytostatic inhibition of cell proliferation. METHODS: Growth inhibition and various regulatory responses were investigated in two human prostate carcinoma cell lines (LNCaP and PC-3) treated with or without a synthetic retinoid, CD 437. RESULTS: Incubation of prostate carcinoma cell lines with a novel retinoid CD437 resulted in the marked inhibition of proliferation. LNCaP and PC-3 possessed IC50 values for CD437 of 375 nM and 550 nM, respectively. Incubation with 1 microM CD437 for 24 hr resulted in 100% and 60% inhibition of growth in LNCaP and PC-3 cells, respectively. Simultaneously, cell flow cytometric analyses revealed a dramatic increase of the cell population in S phase, in both LNCaP (from 38.6% up to 86.7%) and PC-3 (27.9% to 55.7%), and a decreased proportion of cells in G2 phase, in LNCaP (from 23.7% down to 1.2%) and PC-3 (14.9% to 2.2%), indicating a significant S-phase arrest. The cell growth inhibition and S-phase arrest in these cells were followed by apoptosis, as revealed by the acquisition of the characteristic cell morphology including the appearance of apoptotic bodies, and further confirmed by cellular DNA fragmentation. CD437-induced-S phase arrest was associated with upregulated mRNA levels of p21waf1/cip1/sdi1 in both LNCaP (p53+/+) and PC-3 (53-/-) cells. CONCLUSIONS: CD437 represents a unique retinoid that induces S-phase arrest and apoptosis in both androgen-dependent (LNCaP) and -independent (PC-3) human prostate cancer cells, suggesting a potential role of CD437 in the treatment of human prostate cancer.     

The assessment of shoulder instability. The development and validation of a questionnaire

We have developed a 12-item questionnaire for completion by patients presenting with shoulder instability. A prospective study of 92 patients was undertaken involving two assessments, approximately six months apart, performed in an outpatient department. Each patient completed the new questionnaire and the SF36 form. An orthopaedic surgeon completed the Constant shoulder score and the Rowe assessment. The new questionnaire and the Rowe clinical score each achieved a large standardised effect size (> or = 0.8) and compared favourably with relevant items on the SF36. By contrast, the Constant score barely registered any effect, confirming that it may be relatively insensitive to changes in clinical status for this particular condition. The questionnaire provides a measurement of outcome for shoulder instability which is short, practical, reliable, valid and sensitive to changes of clinical importance.     

INT2 and ERBB2 amplification and ERBB2 expression in breast tumors from patients with different outcomes

Summary The relationships of INT2 and ERBB2 amplification and of ERBB2 overexpression in primary breast tumors to prognostic factors, recurrence, and survival have generated considerable controversy. The rationale for this study is that long-term, recurrence-free survival is a more direct criterion for testing the validity of a tumor marker than correlation either with prognostic factors or with short-term recurrence and survival. We examined the association of recurrence with INT2 and ERBB2 amplification and ERBB2 expression by comparing primary breast tumors from patients surviving without recurrence for 8.5 years after diagnosis. the LTS group, to tumors from patients recurring within two years, the RR group. The RR (N = 63) and LTS (N = 61) samples were coded and examined for amplification by Southern blotting and for expression by immunohistochemistry. Comparison between the RR and LTS groups demonstrated that INT2 amplification was associated with a significantly (P = 0.018) higher (5.6-fold) risk of recurrence, an association that remained significant after controlling for lymph node (LN), tumor size (TS), and histograde (HG) status. ERBB2 amplification and expression were not associated with a higher recurrence risk. Survival analyses within the RR group, however, demonstrated significantly shorter survival time among cases with than without ERBB2 amplification (P = 0.018, median survival 16 vs 25 months), or ERBB2 expression (P = 0.019, median survival 15 vs 25 months), but not INT2 amplification. Univariate Cox proportional hazards regression models also demonstrated significantly shorter survival among cases with ERBB2 amplification (P = 0.016) or expression (P = 0.049), that remained significant in multivariate analyses (P = 0.022) for ERBB2 amplification. These results indicate a significant positive association between INT2 amplification and risk for tumor recurrence in the RR as compared to the LTS group. The relationship of ERBB2 amplification or overexpression to patient outcome is more complex. ERBB2 amplification and expression have a significant relationship with shorter survival among patients recurrent within two years, but their occurrence in tumors from women surviving without recurrence for 8.5 years suggests that ERBB2 status is not predictive of shorter survival for all breast cancers.     

Intestinal microsomal drug metabolism. A comparison of rat and guinea-pig enzymes, and of rat crypt and villous tip cell enzymes

A comparison was made of the properties of microsomes prepared from the small intestines of guinea pigs and rats. The NADPH2 cytochrome c reductase activity and cytochrome b5 and cytochrome P-450 content in rat microsomes was 42, 47 and 64% of that in the guinea pig, ethoxycoumarin deethylase activity was comparable, while arylesterase activity was twice as active in rats as guinea pigs. Investigation of the distribution of these and other parameters in rat intestinal epithelia revealed a preferential location of cytochrome P-450 in the villous tip while other parameters showed a more similar distribution between microsomes prepared from the villous tip and crypt.     

A novel neuronal messenger molecule in brain: the free radical, nitric oxide.

Understanding of the organization and function of a newly identified neuronal messenger molecule, nitric oxide, has progressed rapidly. Nitric oxide synthase has been purified and molecularly cloned from brain. Its localization is exclusively neuronal and endothelial. The catalytic activity of nitric oxide synthase accounts for the NADPH diaphorase staining of neurons that are uniquely resistant to toxic insults and neurodegenerative disorders. Nitric oxide has diverse functions. In platelets it inhibits their aggregation, in macrophages it mediates cytotoxicity, and in blood vessels it acts as a vasodilator. In the nervous system nitric oxide may be the retrograde transmitter in long-term potentiation. It is the "neurotransmitter" of cerebral vasodilator nerves and the inhibitory "neurotransmitter" of the motor neurons of the intestines. Nitric oxide in situations of excessive production may function as a neurotoxin, suggesting a role for nitric oxide in neurodegenerative disorders.     

Human acoustic neurinomas: Nervous system specific biochemical parameters

Summary A series of 24 human acoustic neurinomas from 24 patients has been assayed for several biochemical parameters characteristic of the nervous system. S 100 protein, 2, 3-cyclic nucleotide 3-phosphohydrolase activity, and the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide). Myelin basic protein was not detected. These findings further support the neuroectodermal origin of the human acoustic neurinoma, and provide additional biochemical markers for further study.     

Epstein-Barr Virus Polymerase Chain Reaction and Serology in Pediatric Post-Transplant Lymphoproliferative Disorder: Three-Year Experience

To assess whether the semiquantitative peripheral blood Epstein-Barr virus (EBV) polymerase chain reaction (PCR) test correlates with post-transplant lymphoproliferative disorder (LPD), we compiled the results of the test done over a 3-year period ending July 1997. Six hundred seventy-six tests were done on 185 patients. Four hundred-thirty tests (63%) were negative, 167 (25%) were weak positive, 67 (10%) were moderate positive, and 12 (2%) were strong positive. Twelve of the patients developed a lymphoproliferative disorder (LPD) during this time. The EBV PCR tests proximate to the diagnosis of LPD in the 12 patients with EBV-positive LPD were 6 strong positive, 5 moderate positive, 1 weak positive. No patient with LPD had a negative result at diagnosis. Stated another way, 6/12 (50%) of strong-positive PCR tests, 5/67 (7%) moderate-positive tests, and 1/167 (.6%) of weak-positive tests correlated with LPD. Serologic evaluation for EBV done on 7 patients at the time of LPD showed low serologic responses in 5 of the 7 patients. The EBV PCR temporally associated with the serology indicated moderate to large viral burdens. In each patient evaluated serially, the EBV PCR test rose before the diagnosis of LPD and fell with treatment for the disorder. In conclusion, the EBV PCR test may be used as an adjunct to the diagnosis of patients with LPD and may be used to monitor response to therapy for the disorder. Received August 26, 1997; accepted January 13, 1998.     

Growth inhibition of DU-145 prostate cancer cells by a Bcl-2 antisense oligonucleotide is enhanced by N-(2-hydroxyphenyl)all-trans retinamide.

Hormonally insensitive prostate cancer is a relatively slow-growing, but usually fatal, disease with no long-term treatment options. Transformation of normal prostate cells to a malignant phenotype often involves corruption of the apoptotic machineries. Bcl-2 protein is one of the key inhibitors of apoptosis and is often unregulated in advanced prostate cancer. The prostate cancer cell line DU-145 was used as a model of a hormonally insensitive, advanced prostate cancer. Cell growth in liquid culture was significantly inhibited by antisense Bcl-2 oligonucleotides compared with control sense oligonucleotides; inhibition by these oligonucleotides was significantly enhanced on combination with the synthetic retinoid N-(2-hydroxyphenyl)all-trans-retinamide (2-HPR). Interestingly, growth inhibition occurred in the absence of apoptosis as measured using two assay techniques. We hypothesize that in these recalcitrant cells the apoptotic pathway is compromised at several levels, and Bcl-2 may play another role in promoting cell growth. The use of Bcl-2 antisense oligonucleotides plus 2-HPR may provide a novel approach to therapy of hormone-resistant prostate cancer.