Allan Bar-Sinai

The regulation of adenylyl cyclase by receptor-operated G proteins.

The receptor regulated adenylyl cyclase system is a multiprotein complex which is a member of the family of the receptor-effector systems whose signal is transduced by heterotrimeric GTP-binding proteins. The system consists of stimulatory and inhibitory receptors (Rs and Ri), stimulatory and inhibitory G proteins (Gs and Gi) and the adenylyl cyclase enzyme (C). While quite specific in situ, receptors (stimulatory or inhibitory) from one source can activate the appropriate G protein from other cell types or species which in turn can act on C from other sources. Studies with chimeric proteins have shown that the various specificities (stimulatory or inhibitory) can be mapped to defined domains in both receptors and G proteins. The mechanism by which the heterotrimeric G proteins couple to the stimulatory and inhibitory signals is discussed in detail. Specifically, the data supporting collision coupling vs the shuttle mechanism is reviewed, as well as the role of beta gamma subunits in both the stimulatory and inhibitory signals.

Mouse Mammary Tumor Virus Env–Derived Peptide Associates with Nucleolar Targets in Lymphoma, Mammary Carcinoma, and Human Breast Cancer

We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.

The leader peptide of MMTV Env precursor localizes to the nucleoli in MMTV-derived T cell lymphomas and interacts with nucleolar protein B23

We have previously described two nucleolar proteins, named p14 and p21, in MMTV-induced T cell lymphomas. These proteins were identified by a monoclonal antibody (M-66) generated from a nontumorigenic, immunogenic variant of S49 T cell lymphoma. While p14 was common to several MMTV-derived T cell lymphomas, p21 was found only in highly tumorigenic variants of S49 cells. Here we report that p14 is the leader peptide of the MMTV env precursor. The epitope recognized by M-66 contains a putative nuclear localization signal. Actinomycin D was found to induce redistribution of p14/p21 from the nucleolus to the nucleoplasm. p14 coimmunoprecipitated and colocalized with the cellular protein, B23. Association with B23 has been previously reported for other auxiliary nucleolar retroviral proteins, such as Rev (HIV) and Rex (HTLV).

Central nervous system acute lymphoblastic leukemia: role of natural killer cells

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Cranberry Juice Constituents Impair Lymphoma Growth and Augment the Generation of Antilymphoma Antibodies in Syngeneic Mice

In addition to its nutritional value, cranberry juice has been effective in treating urinary tract infections. Various reports have also demonstrated its potential for inhibiting in vitro growth of transformed cell lines. Here we show that a fraction [nondialyzable material (NDM) of a molecular weight range 12,000–30,000 (NDM 12-30K)] derived from cranberry juice impairs in vitro growth and invasion through extracellular matrix of Rev-2-T-6 murine lymphoma cells. Furthermore, intraperitoneal injection of this fraction at nontoxic doses both inhibits the growth of Rev-2-T-6 tumors in vivo and enhances the generation of antilymphoma antibodies. These findings demonstrate the in vivo efficacy of cranberry components against malignant lymphoma in immune competent hosts.

The signal peptide of mouse mammary tumor virus-env: a phosphoprotein tumor modulator.

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers. To look for additional cellular functions of p14, different mutants were ectopically expressed in the MCF-7 human breast cancer cell line. This approach identified motifs responsible for its nucleolar targeting, nucleocytoplasmic shuttling, target protein (B23, nucleophosmin) binding, and phosphorylation at serine 18 and 65 both in situ and in vitro. To test the role of these phosphorylation sites, we carried out in vivo tumorigenesis studies in severe combined immunodeficient mice. The findings show that the p14-Ser65Ala mutation is associated with impaired tumorigenicity, whereas the p14-Ser18Ala mutation is associated with enhanced tumorigenicity. Microarray analysis suggests that phosphorylation at serine 18 or at serine 65 is associated with transcriptional regulation of the L5 nucleolar ribosomal protein (a p14 target) and the Erb-B signal transduction pathway. Taken together, these results show that the phosphorylation status of p14 determines whether it functions as a pro-oncogenic or antioncogenic modulator. Mol Cancer Res; 10(8); 1077–86. ©2012 AACR.

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers.

Reversing ABCB1-mediated multi-drug resistance from within cells using translocating immune conjugates

Multi-drug resistance (MDR) is still a major cause of the eventual failure of chemotherapy in cancer treatment. Different approaches have been taken to render these cells drug sensitive. Here, we attempted sensitizing drug-resistant cells from within, using a translocating immune conjugate approach. To that effect, a monoclonal antibody, C219, directed against the intracellular ATP-binding site of the membrane-anchored MDR transporter ABCB1 [P-glycoprotein (P-gp), MDR1], was conjugated to human immunodeficiency virus [HIV(37–72)Tat] translocator peptide through a disulfide bridge. Fluorescence-labelled IgG-Tat conjugates accumulated in drug resistant Chinese hamster ovary (CHO) cells within less than 20 min. Preincubation with C219-S-S-(37–72)Tat conjugate augmented calcein accumulation in drug-resistant CHO and mouse lymphoma cells, indicating reduction in ABCB1 transporter activity. A thioether conjugate C219-S-(37–72)Tat was ineffective, as were disulfide and thioether conjugates of an irrelevant antibody. Furthermore, in the presence of C219-S-S-(37–72)Tat, drug resistant cells were sensitized to colchicine and doxorubicin. Taken together, these findings demonstrate, as proof of principle, a novel approach for the reversal of MDR from within cells, by delivery of translocating immune conjugates as sensitizing agents towards chemotherapy.

Abstract B30: Signal peptide –mediated immunization against mouse mammary tumor virus (MMTV)-associated tumors

Signal peptides (SPs) are N-terminal extensions on nascent secretory and membrane proteins that mediate insertion into, or translocation across the membrane of the endoplasmic reticulum (ER). A growing number of SPs have been shown to carry out post-ER targeting functions. A case in point is the SP of the envelope precursor protein of Mouse Mammary Tumor Virus (MMTV), a beta retrovirus known to cause lymphoma and mammry carcinoma in laboratory mice. This signal peptide (named MMTV-p14 , or p14 for short) is uniquely localized to nucleoli of cells that harbor the virus where it binds to specific protein targets and functions as a nuclear export factor for viral transcripts. P14 is also a phosphoprotein tumor modulator, the phosphorylation status of which determines whether it will function, in vivo, in a tumor promoting or tumor suppressing capacity. Here we report that p14 is expressed also on the surface of a variety of cells that contain MMTV (murine lymphoma and mammary carcinoma, and human breast cancer cells ectopically expressing p14). Furthermore p14 is immunogenic and vaccination with purified recombinant p14 (using different adjuvants) is sufficient to immunize mice against malignant cells that harbor MMTV. This can be adoptively transferred into naive mice. Furthermore, newly derived monoclonal anti-p14 antibodies with different epitope specificity are now available to study their effect on MMTV associated tumors in vivo as well as for targeted drug delivery to these tumors In view of reports suggesting involvement of MMTV in about 30% of human breast cancers we propose p14 and anti-p14 antibodies as candidates for immunization against MMTV associated malignancies, and as a functional example for other viral signal peptides. Citation Format: Ori Braitbard, Tamar Gross, Maayan Roniger, Allan Bar-Sinai, Nira Hochman, Jacob Hochman. Signal peptide –mediated immunization against mouse mammary tumor virus (MMTV)-associated tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B30.

Abstract A109: A signal peptide as a putative tool against MMTV-associated breast cancers

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma and lymphoma in mice. An increasing body of evidence from different laboratories suggests an association between MMTV and up to 38% of human breast cancers. This seems related to severity of the disease and geographical location. Signal peptides are N-terminal extensions of nascent secretory and membrane proteins (typically including 15-25 amino acid residues). They mediate translocation across, or insertion into the membrane of the endoplasmic reticulum (ER). The signal peptide of Mouse Mammary Tumor Virus (MMTV) envelop precursor (named MMTV-14 or p14 for short), in addition to being unusually long (98 a.a) demonstrates some unique characteristics: 1) It localizes to nucleoli; 2) It functions as a nuclear export factor for intron-containing viral transcripts; 3) It can function in a pro-oncogenic or anti-oncogenic capacity depending on its phosphorylation by the kinases CK2 (at serine 65) or PKC (at serine 18), respectively. We report that, in addition to its nucleolar localization, p14 is expressed on the cell surface of murine lymphomas and mammary carcinomas that harbor MMTV, as well as on the surface of MCF-7 human breast cancer cells ectopically expressing p14. Cell surface expression allows p14 to play the role of a tumor associated antigen. Indeed, priming and boosting of mice with recombinant p14, using different adjuvants, induces specific anti-p14 antibodies, as well as immunizes these mice against malignant tumor cells that harbor MMTV. This immunity is adoptively transferred to naive mice. Thus, MMTV-p14 is a multi-faceted signal peptide with immune modulating characteristics that can be used for developing both preventive and therapeutic approaches towards MMTV-associated breast cancers. Supported in part by a Kamin grant from the Office of the Chief Scientist. Citation Format: Ori Braitbard, Allan Bar-Sinai, Maayan Roniger, Dana Rajchman, Nira Hochman, Jacob Hochman. A signal peptide as a putative tool against MMTV-associated breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A109.