Expression studies have consistently identified tumor protein D52 (TPD52) overexpression in tumor cells. Murine TPD52 (mD52) shares 86% identity with the human orthologue. To study a possible role for TPD52 in transformation, 3T3 fibroblasts were transfected with the full-length cDNA for mD52. Expression of mD52 was confirmed by reverse transcription-PCR (RT-PCR), real-time PCR, and Western blot analysis compared with 3T3 and vector-transfected 3T3 (3T3.V), and the resultant cell line was designated 3T3.mD52. At 4 weeks, 3T3.mD52 gained a 2-fold increase in growth rate, lost contact inhibition, and exhibited a marked phenotype change. Further characterization revealed an acquired ability for anchorage-independent cell growth. To determine whether 3T3.mD52 had become tumorigenic, naïve, healthy, immunocompetent syngeneic mice were inoculated subcutaneously with varying cell doses. Tumors measuring >1 cm 2 were detected 60 days postinoculation with 3T3.mD52, and a 50% subcutaneous tumor incidence was obtained with as few as 5 Â 10 5 3T3.mD52 cells. Remarkably, when lungs from 3T3.mD52 tumor-bearing mice were analyzed, numerous tumor nodules were observed, ranging from nodules less than 10 to nodules too numerous to count (inoculation with 1 Â 10 5 and 5 Â 10 6 cells, respectively). Further support for the metastatic capacity of 3T3.mD52 was the demonstration that transforming growth factor (TGF)-BR1 (receptor) expression decreased and TGF-B1 secretion increased in 3T3.mD52 compared with 3T3 controls. cDNA microarray analysis showed a gene expression pattern that further supported mD52-induced transformation and metastasis. Together, these data suggest that mD52 expression in 3T3 cells initiated cellular transformation, tumorigenesis, and progression to metastasis. (Mol Cancer Res 2007;5(2):133 -44)

Kaposi’s sarcoma (KS) herpesvirus (KSHV) is the etiological agent of several immunodeficiency-linked cancers, including KS. Our previous work showed that the proto-oncogene c-kit is upregulated in KSHV-infected endothelial cells (EC), as well as in KS lesions. We show here that KSHV-dependent induction of both c-kit mRNA and protein requires the establishment of a latent infection and that this upregulation occurs in primary DMVEC as well as in immortalized DMVEC (eDMVEC). Interestingly, we find that while the lymphatic EC (LEC) subpopulation exhibits KSHV-induced c-Kit upregulation, the blood EC (BEC) subpopulation does not. Despite this upregulation of c-Kit, receptor activation and phosphorylation of downstream effectors such as MAP Kinase Erk 1/2 and GSK-3 still requires the addition of exogenous c-Kit ligand, stem cell factor (SCF). These data indicate that KSHV does not induce constitutive c-Kit signaling, but instead upregulates c-Kit receptor levels, thus allowing infected EC to respond to endogenous and exogenous SCF. Nonetheless, inhibition of either c-Kit activation or its downstream effectors reverses the characteristic spindle phenotype of infected eDMVEC. Together, these results contribute to our overall understanding of the role that the c-kit proto-oncogene plays in KS pathogenesis.

Supplementary Data from Induction of Tumorigenesis and Metastasis by the Murine Orthologue of Tumor Protein D52

<div>Abstract<p>Expression studies have consistently identified tumor protein D52 (TPD52) overexpression in tumor cells. Murine TPD52 (mD52) shares 86% identity with the human orthologue. To study a possible role for <i>TPD52</i> in transformation, 3T3 fibroblasts were transfected with the full-length cDNA for <i>mD52</i>. Expression of mD52 was confirmed by reverse transcription-PCR (RT-PCR), real-time PCR, and Western blot analysis compared with 3T3 and vector-transfected 3T3 (3T3.V), and the resultant cell line was designated 3T3.mD52. At 4 weeks, 3T3.mD52 gained a 2-fold increase in growth rate, lost contact inhibition, and exhibited a marked phenotype change. Further characterization revealed an acquired ability for anchorage-independent cell growth. To determine whether 3T3.mD52 had become tumorigenic, naïve, healthy, immunocompetent syngeneic mice were inoculated subcutaneously with varying cell doses. Tumors measuring >1 cm² were detected 60 days postinoculation with 3T3.mD52, and a 50% subcutaneous tumor incidence was obtained with as few as 5 × 10⁵ 3T3.mD52 cells. Remarkably, when lungs from 3T3.mD52 tumor-bearing mice were analyzed, numerous tumor nodules were observed, ranging from nodules less than 10 to nodules too numerous to count (inoculation with 1 × 10⁵ and 5 × 10⁶ cells, respectively). Further support for the metastatic capacity of 3T3.mD52 was the demonstration that transforming growth factor (TGF)-βR1 (receptor) expression decreased and TGF-β1 secretion increased in 3T3.mD52 compared with 3T3 controls. cDNA microarray analysis showed a gene expression pattern that further supported <i>mD52</i>-induced transformation and metastasis. Together, these data suggest that mD52 expression in 3T3 cells initiated cellular transformation, tumorigenesis, and progression to metastasis. (Mol Cancer Res 2007;5(2):133–44)</p></div>

Supplementary Data from Induction of Tumorigenesis and Metastasis by the Murine Orthologue of Tumor Protein D52