Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats.

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

Ubiquitin-proteasome system and increased sensitivity of B-CLL lymphocytes to apoptotic death activation.

The ubiquitin-proteasome-dependent proteolytic system has been reported to regulate apoptotic cell death in many experimental cell models. We recently found that B-CLL (chronic lymphocytic leukemia) lymphocytes are hypersensitive to apoptotic death activation through specific inhibition of proteasome function by lactacystin. Lactacystin efficiently activates apoptotic death process in B-CLL lymphocytes at doses at which no apoptotic effect can be observed in normal human lymphocytes in which 10-fold higher doses of lactacystin are required to weakly induce apoptosis. This hypersensitivity of B-cell CLL may be a result of an altered ubiquitin pathway and proteasomal proteolysis in these malignant cells, and this alteration could be specific for this malignancy. Together with other published works, these results suggest that lactacystin, though not per se a discriminatory inhibitor of the ubiquitinated protein processing/degradation, can nonetheless be discriminatory in the apoptotic cell response between B-CLL and normal lymphocytes: the property that promises efficacy in clinical trials of B-cell CLL. This hypothesis is documented by the fact that lymphocytes from patients in complete remission become resistant to lactacystin-induced apoptosis as normal lymphocytes do.

Chromosomal DNA and p53 stability, ubiquitin system and apoptosis in B-CLL lymphocytes.

The ubiquitin system regulates diverse biological processes such as DNA replication and repair, biogenesis of ribosome, peroxisome and nucleosome, cell cycle, stress response and signal transduction pathways. Thus, the reported role of the ubiquitin system in apoptotic death control as well the alteration of its control in carcinogenesis should come as no surprise. Indeed, we and other groups have reported that the ubiquitin system is involved in apoptotic cell death of normal human lymphocytes and that this control is altered in B lymphocytes derived from chronic lymphocytic leukemia patients (B-CLL), rendering these malignant cells hypersensitive to specific inhibition of protein degradation/processing through proteasomal function. This approach recently allowed us to demonstrate that the stability of the tumor suppressor and pro-apoptotic protein p53 is differentially regulated in B-CLL versus normal lymphocytes and that this difference might at least partly explain the impaired response of B-CLL lymphocytes to apoptotic death activation. These results strongly suggest an imbalance in p53 regulation in B-CLL cells that leads to a variable response to DNA damage and constitutively expressed chromosomal instability. The question we and others would like to address is whether this alteration, or more likely a subset of alterations of the ubiquitin-proteasome pathway, is specific to B-CLL malignancy or if it is a hallmark of cancer cells in general. In either case, a better understanding of the ubiquitin-dependent control of apoptosis should pave the way towards a methodological approach for in vitro development of discriminating treatments which may be of potential usefulness in clinical trials of B-CLL.

High levels of chromosome aberrations correlate with impaired in vitro radiation-induced apoptosis and DNA repair in human B-chronic lymphocytic leukaemia cells.

PURPOSE: To investigate the relationship between the susceptibility of B-chronic lymphoid leukaemia (B-CLL) cells to DNA damage-induced apoptosis, the kinetics of DNA strand-break rejoining, and chromosome damage after exposure to ionizing irradiation. MATERIALS AND METHODS: Lymphocytes from B-CLL patients were gamma-irradiated in vitro with 0.2-5 Gy and stimulated by Staphylococcus aureus cowan I (SAC I) for estimation of chromosomal damage. Induction of apoptosis after irradiation was studied in 50 patients by two methods: morphological characterization of apoptotic cells after fluorescent staining (Hoechst), and specific quantification of mono- and oligonucleosomes in cytoplasmic cell fractions (ELISA assay). Morphological chromosome damage was scored in the first cell generation after irradiation (13 patients). In parallel, the kinetics of DNA single-strand break rejoining were investigated by the alkaline comet assay (12 patients). RESULTS: Ionizing irradiation did not induce apoptosis in lymphocytes from a subset of B-CLL patients. The results suggest that B-CLL cells resistant to radiation-induced apoptosis could repair DNA strand-breaks more rapidly and showed a higher level of chromosome aberrations than radiation-sensitive B-CLL cells. CONCLUSION: Each of three biological effects observed (apoptosis, kinetics of DNA single-strand-break repair, chromosomal damage) might be explained by different modifications occurring in irradiated B-CLL cells. Their convergence strongly suggests that resistance to apoptotic death initiation by DNA damage may be impeded by a rapid engaging of the DNA repair mechanisms. The higher level of chromosome aberrations observed in these cells suggests that the type of DNA repair system involved may generate inaccurate repair.

The tumor suppressor protein PML controls apoptosis induced by the HIV-1 envelope.

Promyelomonocytic leukemia (PML) is a prominent oncosuppressor whose inactivation is involved in the pathogenesis of hematological and epithelial cancers. Here, we report that PML aggregated in nuclear bodies in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. PML aggregation occurred after the fusion of nuclei (karyogamy) within syncytia but before the apoptotic program was activated. The aggregation of PML was detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Using a range of specific inhibitors of PML (the oncogenic PML/RARalpha fusion product or specific small interfering RNAs), we demonstrated that, in Env-elicited syncytia, PML was required for activating phosphorylation of ataxia telangiectasia mutated (ATM), which colocalized with PML in nuclear bodies, in a molecular complex that also involved topoisomerase IIbeta-binding protein 1. PML knockdown thus inhibited the ATM-dependent DNA damage response that culminates in the activation of p53, p53-dependent transcription of pro-apoptotic genes and cell death. Infection of CD4-expressing cells with HIV-1 also induced syncytial apoptosis, which could be suppressed by inhibiting PML. Altogether, these data indicate that PML activation is a critical early event that participates in the apoptotic demise of HIV-1-elicited syncytia.

Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes.

We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific-not nonspecific-proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metabolite clasto-lactacystin beta-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.

Ubiquitin-dependent protein processing controls radiation-induced apoptosis through the N-end rule pathway.

The ubiquitination of nuclear proteins activated in human lymphocytes undergoing radiation-induced apoptosis and the subsequent downstream proteasomal protein processing, shown to be involved in apoptotic death control, may be dependent on an amino-terminal sequence identity of ubiquitin target proteins, the "N-end rule" pathway. Here we report that this selective pathway controls radiation-induced apoptosis and that it is involved in the initiation of this type of cell death. Dipeptide competitors of protein ubiquitination/processing dependent solely on the basic amino-terminal residues (type I) efficiently inhibited the radiation-induced apoptotic death phenotype, indicating that only the substrates of ubiquitination with basic NH2-terminal amino acids are involved in apoptotic death control. This selective inhibition was followed by an early, overall but also target-specific inhibition of ubiquitination and by an activation and stabilization of poly(ADP-ribose) polymerase (PARP) that occurs through inhibition of ubiquitination of its cleaved form (85 kDa). Interestingly, caspases-3 and -7 were not activated following irradiation, further suggesting that PARP cleavage may be regulated by an N-end rule pathway in a caspase-independent manner. These results highly suggest involvement of this subset of the ubiquitin system in the apoptotic death control and in the specific regulation of PARP activity.

Increased sensitivity of CLL-derived lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin.

Ubiquitin-proteasome-dependent protein processing appears to be an essential component in the control of radiation-induced apoptosis in human lymphocytes. This control is altered in chronic lymphocytic leukaemia (CLL), compared to that of normal human lymphocytes which mainly showed high apoptotic values after irradiation, but in some cases no sensitivity was observed. Interestingly, lactacystin activated the apoptotic pathway in both radio-resistant and sensitive CLL cells, at doses which had no effect in normal cells where significantly higher concentrations were required. Therefore the resistance of some CLL cells to apoptosis initiation by radiation does not correlate to observed increased sensitivity to lactacystin. The nuclear level of the transcription factor NF-kappaB or the cytoplasmic level of IkappaBalpha remained unaltered upon irradiation or lactacystin CLL cells treatment, suggesting that the activity of the other factors involved in apoptotic death control were altered through proteasomal inhibition. These results strongly suggest an essential role of the ubiquitin system in apoptotic cell death control in CLL lymphocytes. The inhibition of proteasome-ubiquitin-dependent processing could be a discriminatory apoptotic stimulus between normal versus malignant lymphocytes and therefore might potentially be of use in this specific human pathology.

The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo- and radioresistant human chronic lymphocytic leukaemia lymphocytes to TNF-alpha-initiated apoptosis.

Apoptosis can be triggered by cytotoxic agents and radiation currently used in cancer treatment. However, the apoptotic response appears to vary between cell types (normal or transformed) and between types of malignancy. Thus, irradiation induces apoptosis in normal human lymphocytes but not in lymphocytes derived from a subset of chronic lymphocytic leukaemia (CLL). Moreover, in this subset, spontaneous apoptosis is inhibited by irradiation. Why irradiation does not allow the initiation of the apoptotic death pathway could be explained, at least in part, and in agreement with recent findings on experimental models, by the activation of the transcriptional factor NF-kappaB, which is able to inhibit apoptotic cell response. Low doses (at which no effect is observed with normal human lymphocytes) of the highly specific proteasome inhibitor lactacystin are sufficient to trigger apoptosis in these malignant cells. Proteasome inhibition by lactacystin prevents the nuclear translocation of both p50 and p65 NF-kappaB subunits and sensitizes these cells to apoptosis by tumour necrosis factor (TNF)-alpha treatment. As this subset of CLL is totally resistant to any treatment, proteasome inhibition by lactacystin provides a new therapeutic approach to be explored, considering the sensitivity of malignant CLL-derived lymphocytes to be quite different from that of normal human lymphocytes.