HBZ Protein Supports Proliferation Of Adult T Cell Leukemia Cells Through Suppression Of C/EBPα Signaling

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus etiologically associated with adult T-cell leukemia (ATL). The HTLV-1 bZIP factor (HBZ), which is encoded by minus strand of provirus, plays a critical role in oncogenesis. The HBZ protein is involved in the regulation of multiple signaling associated with tumor, which leads to the human T-cell leukemia. The research showed that HBZ could support the proliferation of ATL cells. However, the precise mechanism of growth promoting activity of HBZ is poorly understood. The C/EBPα signaling plays an important role in cell growth, differentiation, tumorigenesis, immune and so on. C/EBPa acting as a tumor suppressor can suppress the cell growth. In the previous study, it was found that HBZ could interact with C/EBPa, resulting in the impairment of C/EBPa-mediated T cell growth suppression. And like the HBZ protein, C/EBPa also has the bZIP transcription factor. Accumulating evidences show that HBZ involves in the regulation of signaling pathways in ATL by associating with the bZIP transcription factor and affects the various biological processes in the body. So it suggests that HBZ may suppress the C/EBPa transcriptional response by interacted with C/EBPa.In order to investigate why HBZ supports the proliferation of HTLV-1 infected cells, we would reveal the effect of HBZ on the C/EBPa signaling pathway and found the molecular mechanism of growth promoting activity of HBZ on the cellular and molecular levels. The main contents are as follows:Part I:C/EBPa was over-expressed in the adult T-cell leukemiaFirst, it was found that the mRNA level of CEBPA was higher in HTLV-1-infected cell lines (MT-4, MT-1, TL-Oml,43 T-, ED and ATL-2) compared with non-infected one (Jurkat) by real-time PCR. It was confirmed that the protein level of C/EBPa were also higher in ATL cell lines (43 T-and MT-4) by used Western blot. At the same time, It was found that the expression level of CEBPA mRNA was higher in the clinical samples of the six ATL patients than in three healthy donors by real-time PCR. In conclusion, we found that C/EBPa was over-expressed in the adult T-cell leukemia.Part II:HBZ could up-regulate the expression of CEBPA by attributing to the association of HBZ with the CEBPA promoterIn order to study the molecular mechanism of up-regulation of CEBPA in the adult T-cell leukemia, it was firstly found that CEBPA expression level was higher in Kit 225 cell line, which stably express HBZ by real-time PCR. And we also found that the level of CEBPA was high in splenic CD4+T cell from HBZ transgenic mice. Then to analyze the mechanism of up-regulation of CEBPA by HBZ, we cloned the promoter of CEBPA. Moreover, a luciferase assay performed that the promoter activity of CEBPA was dramatically up-regulated by HBZ. And a chromatin immunoprecipitation assay (ChIP) showed that HBZ regulated CEBPA through directly binding the promoter of CEBPA. Taken together, these results collectively indicated that the enhanced induction of CEBPA by HBZ could be attributed to the association of HBZ which the CEBPA promoter.PartⅢ:HBZ repressed C/EBPa-induced transcription through physical association between HBZ and C/EBPa.To clarify the effect of HBZ regulates the C/EBPa signaling pathway, after Jurkat cells were transfected with high expression of HBZ, HBZ could inhibited C/EBPa signaling activation in a dose-dependent manner. To investigate the molecular mechanism by which HBZ suppresses the C/EBPa transcriptional response, it was found that HBZ could physically interact with C/EBPa through an immunoprecipitation assay. In the end, we performed a ChIP assay and found that HBZ could repress the ability of C/EBPα to bind its DNA target. So we could conclude that HBZ repressed C/EBPα-induced transcription through physical association between HBZ and C/EBPα.PartⅣ:HBZ overcomed C/EBPα-mediated suppression of T-cell proliferation through modulating the expression of C/EBPα target genes.To address the molecular mechanism of HBZ affects cell proliferation by suppressing C/EBPα signaling. By using RT-PCR, it was found that HBZ could significantly change the expression of C/EBPα-specific target genes which are associated with cell proliferation. HBZ enhanced E2F1, PCNA, IL6 and suppressed IL4 and IFN-γ in vitro. And it was also found that HBZ could upregulate E2F1, PCNA, IL6 in vivo. Thus, it could conclude that HBZ supports the proliferation of tumor cells through selective modulation of transcription of C/EBPα target genes which are associated with cell proliferation.In conclusion, it was found that HBZ repressed C/EBPα signaling pathway through physical interacting with C/EBPα and diminishing its DNA binding capability. It was firstly showed that HBZ, which is encoded by the HTLV-1 genome, impaired the C/EBPα-mediated the suppression of ATL cells’ growth though modulating the activation of C/EBPα signaling and changing the expression of C/EBPα target genes. This might account for the molecular mechanism of why HBZ supports the proliferation of HTLV-1 infected cells. This study would provide a new insight for expressing carcinogenic mechanism of HTLV-1 and provided a new target for the treatment of adult T-cell leukemia.