Yongdong Feng

Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis

Protein complex of cyclin B1 and cyclin-dependent protein kinase 1 induces phosphorylation of key substrates that mediate cell cycle transition during the G2-M phase. It is believed that cyclin B1 accumulates in the S phase of the cell cycle and reaches the maximal level at mitosis but is absent in G1-phase cells. In the present study, we demonstrated that cyclin B1 was expressed in the arrested G1-phase MOLT-4 lymphocyte leukemia cells and in G1 phase T-7 transitional tumor cells, as determined by flow cytometry. In addition, we showed that cyclin B1 was detected in the G1 phase in breast cancer cells from patient tissues and in lymphocytes from patients with leukemia. These findings were confirmed for the first time by postsorting Western blot analysis and by confocal microscopy. Furthermore, by using postsorting Western blotting, we found that cyclin B1 was expressed in different time-window sections of the G1 phase under different conditions. For the asynchronously growing T-7 cells, cyclin B1 was detected in early G1 phase, whereas in MOLT-4 cells arrested in G1-S phase, cyclin B1 was mainly detected in late G1 phase. We propose that the cyclin B1 expressed in the G1 phase may differ from that expressed in the G2-M phase, and that this unscheduled type of cyclin B1 may play an important role in tumorigenesis and apoptosis.

New method for the analysis of Cell cycle-specific apoptosis

In this study, a new method for the analysis of cell cycle specificity of apoptosis was designed by using a modified annexin V and propidium iodide (API) method.

c-Myc enhances colon cancer cell-mediated angiogenesis through the regulation of HIF-1α.

Angiogenesis plays a pivotal role in tumor growth. The hypoxia-inducible factor 1, α subunit (HIF-1α)/vascular endothelial growth factor pathway is the most important pathway for regulating angiogenesis in the tumor microenvironment. c-Myc is an important oncogene that has many biological functions. In this study, we investigated the role of c-Myc in tumor angiogenesis. We found that the overexpression of c-Myc in colon cancer cells could promote the expression of HIF-1α and that of vascular endothelial growth factor. Moreover, we found that c-Myc regulated HIF-1α at the post-transcriptional level. The results revealed c-Myc-dependent regulation of HIF-1α instead of HIF-1α-dependent c-Myc regulation for the first time. They also showed that c-Myc was essential to regulate colon cancer cell-mediated angiogenesis and contributed to tumor growth. This research provides the theoretical basis for clinical trials of new therapeutic targets of c-Myc and HIF-1α in colon cancer cells.

p55PIK-PI3K stimulates angiogenesis in colorectal cancer cell by activating NF-κB pathway

Vascular growth factor (VEGF) is an important mediator of angiogenesis. PI3K plays essential roles in angiogenesis; however, the mechanisms and specific functions of individual isoforms of PI3K members in tumor angiogenesis regulation are still not fully understood. In this study, we evaluate the role of p55PIK, a PI3K regulatory subunit encoded by PIK3R3 gene, in tumor angiogenesis. We reported that overexpression of p55PIK in cancer cells up-regulated HIF-1α expression and increased VEGF expression. Furthermore, overexpression of p55PIK increased tumor angiogenesis in vivo and in vitro. Moreover, data indicated enhanced HIF-1α expression by p55PIK-PI3K depended on its ability to activate NF-кB signaling pathways, especially to increase the phosphorylation of p65 subunits of NF-κB. Our study suggested that p55PIK-PI3K was essential in regulating cancer cell-mediated angiogenesis and contributed to tumor growth and that the p55PIK provides a potential and specific target for new anti-angiogenesis drug development.

Blocking p55PIK signaling inhibits proliferation and induces differentiation of leukemia cells

p55PIK, a regulatory subunit of phosphatidylinositol 3-kinases, promotes cell cycle progression by interacting with cell cycle modulators such as retinoblastoma protein (Rb) via its unique amino-terminal 24 amino-acid residue (N24). Overexpression of N24 specifically inhibits these interactions and leads to cell cycle arrest. Herein, we describe the generation of a fusion protein (Tat transactivator protein (TAT)–N24) that contains the protein transduction domain and N24, and examined its effects on the proliferation and differentiation of leukemia cells. TAT–N24 not only blocks cell proliferation but remarkably induces differentiation of leukemia cells in vitro and in vivo. Systemically administered TAT–N24 also significantly decreases growth of leukemia cell tumors in animal models. Furthermore, overexpression of p55PIK in leukemia cells leads to increased proliferation; however, TAT–N24 blocks this effect and concomitantly induces differentiation. There is significant upregulation of p55PIK mRNA and protein expression in leukemia cells from patients. TAT–N24 inhibits cell cycle progression and induces differentiation of bone marrow cells derived from patients with several different types of leukemia. These results show that cell-permeable N24 peptide induces leukemia cell differentiation and suggest that p55PIK may be a novel drug target for the treatment of hematopoetic malignancies.

PI3K stimulates DNA synthesis and cell cycle progression via its p55PIK regulatory subunit interaction with PCNA

Previously, we have shown that p55PIK, an isoform of class IA phosphoinositide 3-kinase (PI3K), specifically interacts with important cell-cycle regulators, such as retinoblastoma (Rb), to promote cell-cycle progression. Here, we used the glutathione S-transferase pull-down assay to identify other p55PIK-interacting proteins besides Rb in a Rb-deficient cell line and found that p55PIK interacted with proliferation cell nuclear antigen (PCNA), which plays a key role in coordinating both initiation of the leading strand DNA replication and discontinuous lagging strand synthesis. Overexpression of p55PIK increased, and knockdown decreased, DNA synthesis and DNA replication by modulating the binding of DNA polymerase δ (Polδ) to PCNA. Moreover, a cell-permeable peptide containing the N-terminal–binding domain of p55PIK (TAT–N24) disrupted the p55PIK–PCNA interaction in cancer cells, and also inhibited the DNA synthesis and tumor growth in cell culture and in vivo. Altogether, our results show that the p55PIK–PCNA interaction is important in regulating DNA synthesis and contributes to tumorigenesis. Furthermore, the p55PIK–PCNA interaction provides a potential new target for anticancer drug development. Mol Cancer Ther; 12(10); 2100–9. ©2013 AACR.

Luteinizing Hormone-Releasing Hormone (LHRH)-I Antagonist Cetrorelix Inhibits Myeloma Cell Growth In vitro and In vivo

The objective of this study was to determine the effects of an luteinizing hormone-releasing hormone (LHRH)-I antagonist, Cetrorelix, on human multiple myeloma (MM) cells and to elucidate the mechanisms of action. We showed that LHRH-I and LHRHR-I genes were expressed in MM cell lines and primary MM cells. Treatment with Cetrorelix inhibited growth and colony-forming ability of myeloma cells, including cell lines resistant to arsenic trioxide, bortezomib, or lenalidomide. Cetrorelix induced apoptosis in myeloma cells including primary myeloma cells. In addition, Cetrorelix inhibited the growth of human myeloma cells xenografted into mice without any apparent side effects. Cetrorelix downregulated the nuclear factor-kappa B (NF-κB) pathway activity and the expression of cytokines, including interleukin 6, insulin-like growth factor 1, VEGF-A, and stromal-derived factor 1, important for myeloma cell growth and survival in myeloma cells and/or marrow stromal cells from myeloma patients. Cetrorelix decreased the phosphorylation of extracellular signal regulated kinase 1/2 and STAT3 in myeloma cells, two crucial pathways for myeloma cells growth and survival. Moreover, the expression of p21 and p53 was increased, whereas that of antiapoptotic proteins Bcl-2 and Bcl-xL was reduced by Cetrorelix. Our findings indicate that Cetrorelix induces cytotoxicity in myeloma cells through various mechanisms and provide a rationale for investigating Cetrorelix for the treatment of MM. Mol Cancer Ther; 10(1); 148–58. ©2010 AACR.

Dynamic balance of multiple myeloma clonogenic side population cell percentages controlled by environmental conditions

Cancer stem cells are key drivers of tumor progression and disease recurrence in multiple myeloma (MM). However, little is known about the regulation of MM stem cells. Here, we show that a population of MM cells, known as the side population (SP), exhibits stem‐like properties. Cells that constitute the SP in primary MM isolates are negative or seldom expressed for CD138 and CD20 markers. In addition, the SP population contains stem cells that belong to the same lineage as the mature neoplastic plasma cells. Importantly, our data indicate that the SP and nonside population (NSP) percentages in heterogeneous MM cells are balanced, and that this balance can be achieved through a prolonged in vitro culture. Furthermore, we show that SP cells, with confirmed molecular characteristics of MM stem cells, can be regenerated from purified NSP cell populations. We also show that the percentage of SP cells can be enhanced by the hypoxic stress, which is frequently observed within MM tumors. Finally, hypoxic stress enhanced the expression of transforming growth factor β1 (TGF‐β1) and blocking the TGF‐β1 signaling pathway inhibited the NSP dedifferentiation. Taken together, these findings indicate that the balance between MM SP and NSP is regulated by environmental factors and TGF‐β1 pathway is involved in hypoxia‐induced increase of SP population. Understanding the mechanisms that facilitate SP maintenance will accelerate the design of novel therapeutics aimed at controlling these cells in MM.

Are progenitor cells pre‐programmed for sequential cell cycles not requiring cyclins D and E and activation of Cdk2?

Abstract.  Objectives: Based on studies of unicellular organisms or cultured mammalian cells, the generally accepted model of cell‐cycle regulation has been developed in which sequential (scheduled) expression of cyclins D, E, A and B and activation of Cdk2 and Cdk1 takes place. It is assumed that the same model is applicable both in vivo and in vitro. Materials and methods: In the present study, we compared proliferating marrow cells freshly isolated from healthy individuals with proliferating lymphocytes in cultures. Results: We demonstrate that during progression of freshly collected human bone marrow cells through G1, S and G2/M, only Cdk1 combined with cyclins A and B1 was distinctly present and active, and its activity gradually increased. In contrast, in vitro growing mitogen‐stimulated lymphocytes had perfectly scheduled sequential expression of all four cyclins and Cdk1 and Cdk2 activities. Conclusion: Our findings demonstrate that the pattern of cyclin expression and Cdk activity in bone marrow in vivo is distinctly different from the one observed for normal cells in vitro. Because proliferating bone marrow cells are predominantly expanding populations of committed progenitors, it is likely that during the expansion phase their cell‐cycle progression is pre‐programmed, being driven solely by Cdk1 combined either with cyclin A or with cyclin B1. Expansion of progenitor cells thus may not require the early steps of cell‐cycle regulation, associated with triggering progression by availability of growth factors and mitogens.

DNA damage response in resting and proliferating peripheral blood lymphocytes treated by camptothecin or X-ray.

DNA damage response (DDR) in different cell cycle status of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA). The apoptotic ratio and the phosphorylation H2AX (S139) were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P<0.05). The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.SummaryDNA damage response (DDR) in different cell cycle status of human peripheral blood lymphocytes (PBLs) and the role of H2AX in DDR were investigated. The PBLs were stimulated into cell cycle with phytohemagglutinin (PHA). The apoptotic ratio and the phosphorylation H2AX (S139) were flow cytometrically measured in resting and proliferating PBLs after treatment with camptothecin (CPT) or X-ray. The expressions of γH2AX, Bcl-2, caspase-3 and caspase-9 were detected by Western blotting. DDR in 293T cells was detected after H2AX was silenced by RNAi method. Our results showed that DNA double strand breaks (DSBs) were both induced in quiescent and proliferating PBLs after CPT or X-ray treatment. The phosphorylation of H2AX and apoptosis were more sensitive in proliferating PBLs compared with quiescent lymphocytes (P<0.05). The expression levels of anti-apoptotic proteins Bcl-2 were reduced and cleaved caspase-3 and caspase-9 were increased. No significant changes were observed in CPT-induced apoptosis in 293T cells between H2AX knocking down group and controls. It was concluded that proliferating PBLs were more vulnerable to DNA damage compared to non-stimulated lymphocytes and had higher apoptosis rates. γH2AX may only serve as a marker of DNA damage but exert no effect on apoptosis regulation.