Arsenic trioxide and acute promyelocytic leukemia: clinical and biological.

Arsenic has recently been identified as an effective drug in the treatment of newly diagnosed and relapsed acute promyelocytic leukemia. Indeed, arsenic trioxide combined with all-trans retinoic acid shows a synergistic effect. Mechanistically, arsenic targets the key leukemogenic protein PML-RARalpha, setting up a new example of molecular target-based cancer therapy.

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.

Isolation and characterization of an equine foamy virus.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.

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.

Long-term persistent infection of domestic rabbits by the human foamy virus.

Human foamy virus (HFV) belongs to the spumaretrovirus group of the Retroviridae taxonomic family. Attempts to associate HFV or other foamy viruses to a specific pathology still remain unsuccessful. However, viral gene expression as well as tissue-specific tropism in an in vivo context remain poorly analyzed. To address this issue, we have infected domestic rabbits with a single dose of HFV and followed them at the biological and molecular levels for 5 years. No apparent pathology was detectable in the infected animals which have developed a strong immunological response against major viral proteins. We found that HFV provirus in blood cells and several organs persisted predominantly in its defective form, delta HFV, suggesting that in vivo viral persistence could be related to homologous interference as was recently shown in vitro. This animal model might be useful for studying the in vivo targets of HFV and should also be convenient for testing therapeutic effects of antiretroviral drugs.

The PML and PML/RARalpha domains: from autoimmunity to molecular oncology and from retinoic acid to arsenic.

Acute promyelocytic leukemia (APL) is specifically associated to a t(15; 17) translocation which fuses a gene encoding a nuclear receptor for retinoic acid, RARalpha, to a previously unknown gene PML. The PML protein is localized in the nucleus on a specific domain of unknown function (PML nuclear bodies, NB) previously detected with autoimmune sera from patients with primary biliary cirrhosis (PBC). These bodies are nuclear matrix-associated and all of their identified components (PML, Sp100, and NDP52) are sharply upregulated by interferons. We show that autoantibodies against both PML and Sp100 are usually associated in sera with multiple nuclear dot anti-nuclear antibodies and demonstrate that PML is an autoantigen, not only in PBC, but also in other autoimmune diseases. In APL, the PML/RARalpha fusion interferes with both the retinoic acid (RA) response and PML localization on nuclear bodies, but the respective contribution of each defect to leukemogenesis is unclear. RA induces the terminal differentiation of APL blasts, yielding to complete remissions, and corrects the localization of NB antigens. Arsenic trioxide (As2O3) also induces remissions in APL, seemingly through induction of apoptosis. We show that in APL, As2O3 leads to the rapid reformation of PML bodies. Thus, both agents correct the defect in NB antigen localization, stressing the role of nuclear bodies in the pathogenesis of APL.

Transcriptional induction of the PML growth suppressor gene by interferons is mediated through an ISRE and a GAS element.

PML is a nuclear matrix protein with growth suppressing properties, whose expression is deregulated during oncogenesis. Moreover, in the t(15;17) translocation of acute promyelocytic leukaemia (APL), PML fusion to the retinoic acid receptor alpha (RAR alpha) is the likely molecular basis of leukaemogenesis. Here we show that interferons (IFNs) alpha, beta, and gamma upregulate PML mRNA expression. Analysis of 5' genomic sequences of the PML gene revealed an IFN-alpha/-beta stimulated response element (ISRE) and an IFN-gamma activation site (GAS) in the untranslated first exon. Binding of IFN signal transducers and activators of transcription (STATs) was demonstrated to be weak for the PML GAS, but strong for the PML ISRE which also seemed to contribute substantially to the IFN-gamma response. Thus, PML is a primary target gene of IFNs and would appear as a suitable candidate for mediating some of their antiproliferative effects. Abnormalities of PML structure, localisation or expression in human malignancy, constitute examples of how an IFN target gene may be altered in oncogenesis.

Sequestration of PML and Sp100 proteins in an intranuclear viral structure during herpes simplex virus type 1 infection.

We investigated the intranuclear distribution of PML and Sp100 in HeLa cells at the ultrastructural level and examined their relocalization in response to herpes simplex virus type 1 (HSV-1) infection. In the absence of infection, we observed that both are components, not only of nuclear bodies, but also of interchromatin granule-associated zones, which suggests a potential role for PML and Sp100 in splicing events. Prolonged HSV-1 infection induced dramatic changes in nuclear organization which consisted of the morphological disappearance of some nuclear structures (nuclear bodies, interchromatin granule-associated zones, coiled bodies) and of the development of a centrally located electron-translucent viral region which pushed the cellular clusters of interchromatin granules to the nuclear border. Concomitantly, dense bodies, concentric arrays of reduplicated inner nuclear membrane, and translucent patches containing a few viral capsids occurred at the nuclear border. PML and Sp100 were exclusively detected over the finely granular material of the viral translucent patches which also contains small amounts of p80-coilin and U1 and U2 snRNAs. An antiserum raised against capsid proteins intensely labeled the viral translucent patches at the level of their finely granular material and enclosed viral capsids. Our data, therefore, suggest that these viral structures, in addition to being the site of accumulation of viral capsid proteins and, possibly, a capsidworks, are also a site of sequestration of cell factors including PML and Sp100. Viral capsid proteins could interfere with and inactivate PML and Sp100 and be implicated in the shutoff of host cell metabolism induced by HSV-1 infection.

The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.

Nuclear bodies (NBs) are ultrastructurally defined granules predominantly found in dividing cells. Here we show that PML, a protein involved in the t(15;17) translocation of acute promyelocytic leukaemia (APL), is specifically bound to a NB. PML and several NB-associated proteins, found as auto-antigens in primary biliary cirrhosis (PBC), are co-localized and co-regulated. The APL-derived PML-RAR alpha fusion protein is shown to be predominantly localized in the cytoplasm, whereas a fraction is nuclear and delocalizes the NB antigens to multiple smaller nuclear clusters devoid of ultrastructural organization. RA administration (which in APL patients induces blast differentiation and consequently complete remissions) causes the re-aggregation of PML and PBC auto-antigens onto the NB, while PML-RAR alpha remains mainly cytoplasmic. Thus, PML-RAR alpha expression leads to a RA-reversible alteration of a nuclear domain. These results shed a new light on the pathogenesis of APL and provide a molecular link between NBs and oncogenesis.

PML protein expression in hematopoietic and acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is thought to be caused by the t(15,17) translocation that fuses the PML gene to that of the retinoic acid receptor alpha (RAR alpha) and generates a PML/RAR alpha fusion protein. Yet, paradoxically, APL cells are exquisitely sensitive to retinoic acid (RA), as they terminally differentiate upon RA exposure. In this report, we have examined the expression of PML and PML/RAR alpha in normal and APL cells. By immunofluorescence or immunocytochemistry, we show that PML has a speckled nuclear pattern of expression that contrasts with that of PML/RAR alpha (mostly a micropunctuated nuclear pattern or a cytoplasmic localization). The APL-derived cell line NB4 that expresses both the PML and PML/RAR alpha genes also shows the fine micropunctuated nuclear pattern, suggesting a dominant effect of the fusion protein over the localization of wild-type PML. RA treatment of NB4 cells or clones expressing PML/RAR alpha gradually leads to a PML pattern before apparent morphologic maturation. In 14 untreated APL patients, the PML-reactive proteins were cytoplasmic (by immunocytochemistry) or both cytoplasmic and nuclear with a micropunctuated pattern (by immunofluorescence). Strikingly, in 4 patients, after 1 to 2 weeks of RA therapy, the speckled nuclear PML pattern reappeared concomitant with the onset of differentiation. These results establish that fusion of PML to RAR alpha results in an altered localization of PML that is reverted upon RA treatment. This observation, which highlights the importance of PML, is likely to be a key to unravelling the molecular mechanism of both leukemogenesis and RA-induced differentiation of APL.

Human foamy virus polypeptides: identification of env and bel gene products.

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product.

First heterotransplantation of a human carcinoid tumor into nude mice.

The first successful heterotransplantation of a human carcinoid tumor into nude mice is reported. CSH, a voluminous hepatic metastasis of a primary bronchial carcinoid tumor (CSB) was resected and transplanted into three irradiated nude (Swiss-nu/nu) mice both by subcutaneous (SC) and intramuscular (IM) routes; the success rate was five of six. Heterotransplanted tumors took 4 to 5 months to appear in the mice and 1 month to attain a width of 0.5 cm. Both human and mouse tumors (named CSH-SC and CSH-IM) were studied by light and electron microscopy. They were Grimelius-positive, neuron-specific enolase-positive, and bombesin-negative by immunocytochemistry. Furthermore, CSH-SC cells presented characteristic (pear-shaped, rod-shaped, or tadpole-shaped) neurosecretory granules. Although CSB and CSH were slightly serotonin positive by immunocytochemistry, only a few serotonin-positive cells were found in CSH-SC and none in CSH-IM, suggesting partial loss of differentiation or an increase in serotonin catabolism during transplantation.

[Production of a monoclonal antibody (B1N) recognizing human nuclear antigen associated with cell proliferation]. / Production d'un anticorps monoclonal (B1N) reconnaissant un antigène nucléaire humain lié à la prolifération cellulaire.

This report describes the preliminary characterization of a novel antigen reactive with a murine monoclonal antibody designated B1N produced in our laboratory. This antibody (IgM) reacts in IFI with mammals and also insect cells, by staining in a speckled fashion the nucleus of these cells. Immunoblotting analysis of Hela and murine D55 nuclear extracts revealed a polypeptide with an apparent molecular weight of 120kD (p120). In this work we demonstrated that: 1. this polypeptide appeared in human peripheral blood lymphocytes only when they were induced to proliferate in vitro after phytohemagglutinin stimulation; 2. this polypeptide was no longer detected in D55 resting cells, following serum deprivation; 3. the MAb B1N specifically revealed the nucleus of proliferating cells on frozen sections of uterine tissue. These data strongly suggest that the p120 nuclear antigen expression is associated with the proliferation state of cells.

Molecular differences between two immunologically related spumaretroviruses: the human prototype HSRV and the chimpanzee isolate SFV6.

A comparative study at the genomic and protein level was performed between two immunologically related spumaretroviruses, the human HSRV and the simian SFV6. Cross immunoprecipitation analysis with specific polyclonal and monoclonal antisera indicates shared antigenic determinants. However, restriction analysis of the viral DNAs and thermal stability of the hybrids demonstrate that HSRV and SFV6 are two different isolates.

Effect of recombinant human alpha A interferon on Visna virus multiplication in acutely infected ovine cells.

We have studied the effects of recombinant human alpha A interferon (IFN) on the multiplication of Visna retrovirus in ovine cells. Pretreatment with IFN followed by continuous IFN treatment, drastically blocked the Visna virus induced cytopathogenic effect and the emergency of neoformed virions. Thus, virus yield and virus dependent reverse transcriptase activity were highly reduced in supernatant fluids. All these effects were accompanied by a strong inhibition of viral protein synthesis. In the light of these data, human IFN action on Visna retrovirus seems to be determined by a mechanism of action distinct from that described in the case of other Retroviridae.

A novel monoclonal antibody (7B10) with differential reactivity between human mammary carcinoma and normal breast.

A monoclonal antibody (7B10) which displays differential reactivity with breast carcinomas compared to benign lesions or normal breast tissue was selected by fusion of spleen cells from BALB/c mice immunized with the T47D human mammary carcinoma cell line. The antigen, recognized by 7B10 on T47D cells, appeared to be both surface and cytoplasm localized, as demonstrated by indirect immunofluorescence, immunoperoxidase, and electron microscopy studies. This antibody (IgG1) bound with four human breast cancer cell lines (T47D, MCF7, ZR-75-1, and HSL53) which express estrogen receptors. No binding was observed with cancer cell lines of other origin or with normal cells. In vivo, by immunoperoxidase staining of frozen sections of normal breast, the antigen recognized by 7B10 appeared to be located on epithelial cell membranes, whereas in benign and malignant mammary disorders, staining also involved the cytoplasm, as confirmed by electron microscopy on fresh cancer tissue. On formalin-fixed, paraffin-embedded sections, cytoplasmic staining was detected in breast cancer, but no immunostaining was observed with benign lesions or normal breast. In paraffin sections, most normal tissues investigated did not react with 7B10 antibody. However, ducts in the parotid gland, tubules in the kidney and some biliary ducts, and apocrine glands in the skin showed irregular, diffuse weak staining. 7B10 was unreactive with adenocarcinomas of origin other than breast, except for some cells in ovarian clear cell carcinoma. No reactivity was observed with squamous carcinomas, lymphomas, or melanomas. The antigen recognized by 7B10 appeared to be a Mr 32,000 protein, as identified by immunoprecipitation from extracts of T47D after labeling with [35S]methionine. Since the antigen was present only on the membrane of differentiated normal mammary epithelial cells, and was also expressed in the cytoplasm of tumor cells, it may be of interest in immunological studies of mammary epithelial cell differentiation. Moreover, since in formalin-fixed tissues immunostaining is virtually confined to mammary carcinomas, monoclonal antibody 7B10 may have diagnostic applications in breast cancer.

Application of an in vivo culture system for rapid demonstration of anti-Leishmania activity of monoclonal antibodies.

In mice, infection with Leishmania by the subcutaneous route becomes evident after about 2 months. This delay impedes the selection of monoclonal antibodies able to interfere with the infectiousness of the parasite. Using an in vivo culture system--intraperitoneal injection of TG 180 sarcoma cells along with promastigotes or amastigotes--it was possible to define within 15 to 20 days a monoclonal antibody preventing the development of Leishmania. Pretreatment of promastigotes and amastigotes of several Leishmania species with a monoclonal antibody raised against Leishmania infantum prevented infection equally in either system. These cross-reactions may be of importance in designing new approaches of immunotherapy.

[Monoclonal antibodies directed against breast cancer associated antigens]. / Anticorps monoclonaux dirigés contre des antigènes associés aux cancers mammaires.

Two mouse hybridomas, producing the monoclonal antibodies 7B10 and 1BE12 which react with membrane antigens of a metastatic human breast tumor cell line were selected. One of them, 7B10, is directed against a mammary gland antigenic determinant and selectively stain mammary carcinoma on histologic sections after fixation and paraffin embedding.

Monoclonal antibodies to murine retrovirus protein p30.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of Wistar-Furth rats that had been immunized with a Moloney sarcoma virus (Mo-MuSV)-induced tumour, MFU. Two immunization protocols were designed. In the first, the animals received several injections of irradiated (10 000 rad) cells of a tumour cell line established in vitro, MFU-67. The rats received a booster injection 3 days prior to fusion. In the second protocol, immunization was the result of simple tumour growth, and no booster was given. Hybrids were tested by immunofluorescence for the production of immunoglobulins reacting with mouse cells acutely infected with Mo-MuSV. Over 20% of reactive hybrids were observed in the tumour growth protocol, and about 10% in the irradiated cell protocol when the last injection of the series was given 2 weeks before fusion. After 6 months, the proportion fell to 3%. Hybrid lines producing antibody to p30, the major core polypeptide of murine retroviruses, were obtained by cloning. Three of these were selected for closer study and were found to recognize three non-overlapping epitopes on p30. By direct and competitive binding in ELISA tests, the three epitopes were found to have very different distribution patterns among the various strains and isolates of murine retroviruses.