Pathways of retinoic acid- or arsenic trioxide-induced PML/RARalpha catabolism, role of oncogene degradation in disease remission.

Although there is evidence to suggest that PML/RARalpha expression is not the sole genetic event required for the development of acute promyelocytic leukemia (APL), there is little doubt that the fusion protein plays a central role in the initiation of leukemogenesis. The two therapeutic agents, retinoic acid and arsenic, that induce clinical remissions in APL, both target the oncogenic fusion protein, representing the first example of oncogene-directed cancer therapy. This review focuses on the molecular mechanisms accounting for PML/RARalpha degradation. Each drug targets a specific moiety of the fusion protein (RARalpha for retinoic acid, PML for arsenic) to the proteasome. Moreover, both activate a common caspase-dependent cleavage in the PML part of the fusion protein. Specific molecular determinants (the AF2 transactivator domain of RARalpha for retinoic acid and the K160 SUMO-binding site in PML for arsenic) are respectively implicated in RA- or arsenic-triggered catabolism. The respective roles of PML/RARalpha activation versus its catabolism are discussed with respect to differentiation or apoptosis induction in the context of single or dual therapies.

PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator.

The promyelocytic leukaemia (PML) protein localizes in the nucleus both in the nucleoplasm and in matrix-associated multiprotein complexes known as nuclear bodies (NBs). The number and the intensity of PML NBs increase in response to interferon (IFN). Overexpression of PML affects the replication of vesicular stomatitis virus and influenza virus. However, PML has a less powerful antiviral activity against these viruses than the IFN mediator MxA. Here, we show that overexpression of PML, but not that of Mx1 or MxA, leads to a drastic decrease of a complex retrovirus, the human foamy virus (HFV), gene expression. PML represses HFV transcription by complexing the HFV transactivator, Tas, preventing its direct binding to viral DNA. This physical interaction requires the N-terminal region of Tas and the RING finger of PML, but does not necessitate PML localization in NBs. Finally, we show that IFN treatment inhibits HFV replication in wild-type but not in PML-/- cells. These findings point to a role for PML in transcriptional repression and suggest that PML could play a key role in mediating an IFN-induced antiviral state against a complex retrovirus.

Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation.

Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) alpha expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARalpha and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARalpha degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation.

BCL-2 cooperates with promyelocytic leukemia retinoic acid receptor alpha chimeric protein (PMLRARalpha) to block neutrophil differentiation and initiate acute leukemia.

The promyelocytic leukemia retinoic acid receptor alpha (PMLRARalpha) chimeric protein is associated with acute promyelocytic leukemia (APL). PMLRARalpha transgenic mice develop leukemia only after several months, suggesting that PMLRARalpha does not by itself confer a fully malignant phenotype. Suppression of apoptosis can have a central role in tumorigenesis; therefore, we assessed whether BCL-2 influenced the ability of PMLRARalpha to initiate leukemia. Evaluation of preleukemic animals showed that whereas PMLRARalpha alone modestly altered neutrophil maturation, the combination of PMLRARalpha and BCL-2 caused a marked accumulation of immature myeloid cells in bone marrow. Leukemias developed more rapidly in mice coexpressing PMLRARalpha and BCL-2 than in mice expressing PMLRARalpha alone, and all mice expressing both transgenes succumbed to leukemia by 7 mo. Although both preleukemic, doubly transgenic mice and leukemic animals had abundant promyelocytes in the bone marrow, only leukemic mice exhibited thrombocytopenia and dissemination of immature cells. Recurrent gain of chromosomes 7, 8, 10, and 15 and recurrent loss of chromosome 2 were identified in the leukemias. These chromosomal changes may be responsible for the suppression of normal hematopoiesis and dissemination characteristic of the acute leukemias. Our results indicate that genetic changes that inhibit apoptosis can cooperate with PMLRARalpha to initiate APL.

Comparison between hybrid capture II and polymerase chain reaction results among women at low risk for cervical cancer.

PURPOSE: Hybrid Capture (HC) is a new and powerful tool to detect DNA/RNA of several infectious agents. Regarding HPV DNA detection HC II has shown no false positive results due to contamination and its inter-laboratory reproducibility reaches 98%. It is also simpler and costs less than other methods such as Polymerase Chain Reaction (PCR) and it has shown a high correlation to clinical findings. Our objective is to compare HC II and PCR results among women at low risk for cervical cancer.METHODS: This is a cross-sectional study which enrolled 977 asymptomatic women aged 15 and 70 years. Squamous columnar junction samples were obtained and analyzed for HPV DNA either by HC II and PCR. Epidemiological factors and cyto-pathologic results were related to HPV DNA status. The Pearson Chi-square test and multiple logistic regression were performed to relate epidemiological and cytopathological variables and HPV DNA status.RESULTS: About 15.4% and 15.9% of women were HPV DNA positive by HC II and PCR respectively. Both were highly associated with cytology (P < 0.0001). The correlation to histopathology was also good with a higher significance for HC II. Factors related to HPV DNA detection by PCR were: oral contraceptive use (OR = 1.26; 95% CI = 1.2-1.62); history of genital HPV infection (2.11; 1.13-4.59); 3 or more sexual partners (1.35; 1.10-1.83); education (

Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia.

In acute promyelocytic leukemia (APL) patients, retinoic acid (RA) triggers differentiation while arsenic trioxide (arsenic) induces both a partial differentiation and apoptosis. Although their mechanisms of action are believed to be distinct, these two drugs both induce the catabolism of the oncogenic promyelocytic leukemia (PML)/RARalpha fusion protein. While APL cell lines resistant to one agent are sensitive to the other, the benefit of combining RA and arsenic in cell culture is controversial, and thus far, no data are available in patients. Using syngenic grafts of leukemic blasts from PML/RARalpha transgenic mice as a model for APL, we demonstrate that arsenic induces apoptosis and modest differentiation, and prolongs mouse survival. Furthermore, combining arsenic with RA accelerates tumor regression through enhanced differentiation and apoptosis. Although RA or arsenic alone only prolongs survival two- to threefold, associating the two drugs leads to tumor clearance after a 9-mo relapse-free period. These studies establishing RA/arsenic synergy in vivo prompt the use of combined arsenic/RA treatments in APL patients and exemplify how mouse models of human leukemia can be used to design or optimize therapies.

SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation.

PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1. We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.

Contraceptive practices of patients referred for colposcopy with an abnormal cervical smear.

This study examined the contraceptive practices of 100 patients referred for colposcopy because of an abnormal cervical smear. At presentation 49% were using oral contraception but 94% had taken the pill at some time. Eight of the 10 patients using barrier methods were relying on condoms: in at least seven of the eight cases an abnormal cervical smear preceded condom usage. Thus, the method of contraception used when cervical neoplasia develops may differ from the method used when the patient presents for colposcopy. Although consistent with previous studies suggesting that the incidence of cervical neoplasia is increased in women using oral contraception, the risk of neoplasia is more likely to be explained by the degree of exposure of the cervix to an infectious carcinogen or to the immunosuppressive effects of seminal plasma. We suggest that future studies of the epidemiology and natural history of cervical neoplasia should include a detailed contraceptive history.