Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties.

Here we report the identification of a new insulin-like growth factor binding protein homologue, provisionally designated insulin-like growth factor binding related protein-4 (IGFBP-rP4). IGFBP-rP4 was found to be most closely related to IGFBP-7 with 52% amino acid homology and 43% amino acid identity, and shares a similar domain structure. Semi-quantitative RT-PCR expression analysis demonstrated a pattern of downregulation of this gene in multiple tumor samples including lung and colon cancer, compared to matched adjacent normal tissue. Western blotting revealed a protein of approximately 38kDa expressed in both the cell pellet and secreted into the supernatant of transiently transfected Cos-7 cells. Cos-7 supernatants containing IGFBP-RP4 protein were observed to suppress the growth of HeLa cells in culture compared to vector controls. IGFBP-RP4 directly transiently transfected into HeLa cells also further confirmed the growth suppressive properties of this protein. Together these data suggest that IGFBP-RP4 may be a novel putative tumor suppressor protein.

The development of a model for the homing of multiple myeloma cells to human bone marrow.

Prior in vitro studies have suggested a role of adhesion molecules, bone marrow stromal cells (BMSCs), and cytokines in the regulation of human multiple myeloma (MM) cell growth and survival. Although in vivo models have been developed in severe combined immunodeficient (SCID) mice that support the growth of human MM within the murine BM microenvironment, these xenograft models do not permit a study of the role of adhesion proteins in human MM cell-human BMSC interactions. We therefore established an in vivo model of human MM using SCID mice implanted with bilateral human fetal bone grafts (SCID-hu mice). For the initial tumor innoculum, human MM derived cell lines (1 x 10(4) or 5 x 10(4) ARH-77, OCI-My5, U-266, or RPMI-8226 cells) were injected directly into the BM cavity of the left bone implants in irradiated SCID-hu mice. MM cells engrafted and proliferated in the left human fetal bone implants within SCID-hu mice as early as 4 weeks after injection of as few as 1 x 10(4) MM cells. To determine whether homing of tumor cells occurred, animals were observed for up to 12 weeks after injection and killed to examine for tumor in the right bone implants. Of great interest, metastases to the right bone implants were observed at 12 weeks after the injection of 5 x 10(4) MM cells, without spread of human MM cells to murine BM. Human MM cells were identified on the basis of characteristic histology and monoclonal human Ig. Importantly, monoclonal human Ig and human interleukin-6 (IL-6), but not human IL-1beta or tumor necrosis factor-alpha, were detectable in sera of SCID-hu mice injected with MM cells. In addition, specific monoclonal Ig light chain deposition was evident within renal tubules. This in vivo model of human MM provides for the first time a means for identifying adhesion molecules that are responsible for specific homing of human MM cells to the human, as opposed to murine, BM microenvironment. Moreover, induction of human IL-6 suggests the possibility that regulation of MM cell growth by this cytokine might also be investigated using this in vivo model.

Interleukin-6 is required for pristane-induced plasma cell hyperplasia in mice.

Intraperitoneal injection of pristane induces production of interleukin-6 (IL-6) and either plasmacytosis or plasmacytoma in mice, depending upon the genetic background. Pristane does not induce plasmacytoma in IL-6 knockout (IL-6-/-) mice, suggesting that IL-6 is required for this process. In the present study we determined whether IL-6 is also required for pristane-induced hyperplasia of normal plasma cells. Pristane was injected intraperitoneally into IL-6-/- and IL-6 wild-type (IL-6+/+) mice. Overall there were more deaths in IL-6+/+ mice (85%) than in IL-6-/- mice (40%), P = 0.024. Hyperplastic lymph node and spleen weight did not differ (P = 0.82 and P = 0.15, respectively) in IL-6-/- versus IL-6+/+ mice. Lymphocytosis with similar patterns of expression of B-cell (B220) and T-cell (Thy-1) antigens was noted in both IL-6-/- and IL-6+/+ mice. However, morphological studies, dual fluorescent staining for Syn-1 and B220 antigens (syn-1+ B220+ cells), and intracytoplasmic Ig staining revealed plasma cell hyperplasia in lymph node and spleen from IL-6+/+, but not IL-6-/-, mice. These plasma cells from IL-6+/+ mice were polyclonal and unable to induce tumour formation in severe combined immunodeficient mice. These data demonstrate that IL-6 is required for pristane-induced hyperplasia of polyclonal plasma cells in mice.

Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells.

Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF-beta1) is also produced by BMSCs and can regulate IL-6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF-beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N-BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N-BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM-BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF-beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion.

Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.

Chimeric homeobox gene E2A-PBX1 induces proliferation, apoptosis, and malignant lymphomas in transgenic mice.

Expression of the homeobox fusion gene E2A-PBX1 under control of the immunoglobulin heavy chain enhancer efficiently induced malignancies in transgenic mice. All animals died before 5 months of age with lymphomas that demonstrated phenotypes consistent with transitional intermediate thymocytes (CD4+/CD8+/CD3med). E2A-PBX1 also markedly altered lymphoid development in pretumorous animals, reducing the number of thymocytes and bone marrow B lineage progenitors to 20% of normal levels. In spite of the observed reductions in lymphoid cells, premalignant animals contained significantly increased numbers of cycling thymocytes, but a higher proportion was also undergoing apoptosis, suggesting that increased cell death resulted in the marked lymphopenias. These data indicate that the chimeric homeodomain protein E2A-PBX1 paradoxically induces both proliferation and apoptosis in lymphoid cells, suggesting an in vivo association between nuclear oncogene-induced cell cycle progression and programed cell death.

Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function.

Transmembrane envelope protein (TM) residues 100, 105, and 128 of human immunodeficiency virus type 1 (HIV-1) strain HXB2 are potential sites for asparagine-linked oligosaccharide additions which are conserved among HIV-1 isolates, and all other lentivirus TM proteins. Site-specific mutants of each of the asparagine residues did not eliminate the ability of the virus to infect and replicate in CD4+ cells, but infectivity was reduced with all of these mutants, and syncytia induction was attenuated with two of these mutants. Studies of envelope expression of the mutant with the most severe defect demonstrated no significant effects on envelope protein synthesis, conformation, processing, multimerization, or release into the culture medium, suggesting that N-linked oligosaccharides are important in the specific fusion activity of TM.