Jianping Gong

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.

G1-phase specific apoptosis in liver carcinoma cell line induced by copper-1,10-phenanthroline

Reactive oxygen species play an important role in the mediation of cell killing. But the mechanistic links between reactive oxygen species (ROS) and cell death remains unclear. There was a speculation that ROS, especially hydroxyl radicals can induce necrosis but not apoptosis in cells treated with copper-1,10-phenanthroline, IICu(OP)(2). In this paper, liver carcinoma cell line (Bel-7402) was treated with IICu(OP)(2) and its effect was examined by several means. Cells were found to undergo changes characteristic of apoptosis. Hoechst staining showed apoptotic body appeared in the cells induced by IICu(OP)(2). When DNA extracted from the cells treated with IICu(OP)(2) was analyzed by agarose gel electrophoresis it generated ladder pattern of discontinuous DNA fragments. Sub-G(1) peak was detected in treated cells. Furthermore, two different flow cytometric methods were used, each allowing us to relate the apoptotic cells to the position the cell-cycle position. Apoptosis induced by IICu(OP)(2) was limited to G(1)-phase cells. Using cyclin analysis, the expression of cyclin E in G(1) was blocked. Thus, it was concluded that IICu(OP)(2) can induce G(1)-phase specific apoptosis in Bel-7402.

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.

p55PIK Transcriptionally Activated by MZF1 Promotes Colorectal Cancer Cell Proliferation

p55PIK, regulatory subunit of class IA phosphatidylinositol 3-kinase (PI3K), plays a crucial role in cell cycle progression by interaction with tumor repressor retinoblastoma (Rb) protein. A recent study showed that Rb protein can localize to the mitochondria in proliferative cells. Aberrant p55PIK expression may contribute to mitochondrial dysfunction in cancer progression. To reveal the mechanisms of p55PIK transcriptional regulation, the p55PIK promoter characteristics were analyzed. The data show that myeloid zinc finger 1, MZF1, is necessary for p55PIK gene transcription activation. ChIP (Chromatin immuno-precipitation) assay shows that MZF1 binds to the cis-element “TGGGGA” in p55PIK promoter. In MZF1 overexpressed cells, the promoter activity, expression of p55PIK, and cell proliferation rate were observed to be significantly enhanced. Whereas in MZF1-silenced cells, the promoter activity and expression of p55PIK and cell proliferation level was statistically decreased. In CRC tissues, MZF1 and p55PIK mRNA expression were increased (P = 0.046, P = 0.047, resp.). A strong positive correlation (Rs = 0.94) between MZF1 and p55PIK mRNA expression was observed. Taken together, we concluded that p55PIK is transcriptionally activated by MZF1, resulting in increased proliferation of colorectal cancer cells.

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.

hSav1 interacts with HAX1 and attenuates its anti-apoptotic effects in MCF-7 breast cancer cells.

It has been reported that Salvador (SAV) is a core component of the Salvador-Warts-Hippo (SWH) pathway that restricts cell number, by functioning as a dual regulator of cell proliferation and apoptosis in Drosophila. However, the function of its human ortholog hSav1 (also called hWW45) in mammalian cells is poorly understood. In this study, we identified hematopoietic cell-specific protein 1 (HS1)-associated protein X-1 (HAX1), a 35-kDa protein localized to cell mitochondria, as a novel binding partner of hSav1 using a yeast two-hybrid screening technique. Our finding was confirmed by immunoprecipitation and glutathione-S-transferase (GST) pull-down assays of both proteins. Using immunofluorescence staining, we showed that HAX1 and hSav1 interact with each other. Analysis of the anti-apoptotic function of HAX1 revealed that the presence of hSav1 attenuated the HAX1 protective effects from hydrogen peroxide (H2O2)-induced cell death in MCF-7 cells, while knockdown of hSav1 by small interfering RNAs (siRNAs) significantly enhanced the anti-apoptotic function of HAX1. Also, using the Oncomine database, we found several studies in which HAX1 levels were significantly up-regulated and hSav1 expression was down-regulated in breast cancer samples compared to normal breast tissue. In summary, we conclude that hSav1 interacts with HAX1 and attenuates its protective role against apoptosis in MCF-7 breast cancer cells.

Mesogastrium: a fifth route of metastasis in gastric cancer?

Radical gastrectomy for gastric cancer with D2 lymph node dissection has been widely applied in advanced gastric cancer. It is believed that such surgery should extremely sweep away local-regional tumor tissues and cancer cells and thoroughly prevent tumor recurrence in situ. However, for most patients with advanced gastric cancer, tumor local-regional recurrence has been proven unavoidable. This study has found that isolated cancer cells, separate from the primary lesion and lymph nodes, existed in the mesogastrium of resected gastric cancer specimens, leading to the hypothesis that these cancer cells may have infiltrated the mesogastrium through a fifth metastasis route (here named Metastasis V) which is distinct from the other four classic metastasis routes, and cannot be resected by conventional radical gastrectomy with D2 lymph node dissection. Local-regional recurrence might be closely associated with these cancer cells in the mesogastrium, and therefore, complete mesogastrium excision (CME) should be imperative and become the third radical principle for radical gastrectomy.

Role of RANTES and its receptor in gastric cancer metastasis.

This study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.SummaryThis study examined the role of regulated upon activation normal T cell expressed and secreted (RANTES) and its receptor C-C chemokine receptor type 5 (CCR5) in gastric cancer metastasis and the associated mechanism. The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis (n=30 in each). The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis (P<0.05). The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes (P<0.05). Chemotactic test revealed that the number of migrating gastric cancer cells (n=295.0±54.6) induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody (n=42.5±11.6) (P<0.05). RT-PCR showed that the expression levels of the main Th1 cytokines (IL-2, Γ-IFN) were lower in gastric cancer with lymph node metastasis (2.22±0.90, 3.26±1.15 respectively) than in that without metastasis (3.07±1.67, 4.77±1.52 respectively) (P<0.05), but the expression level of the main Th 2 cytokine (IL-10) was higher in gastric cancer with lymph nodes metastasis (6.06±2.04) than in that without metastasis (4.88±1.87) (P<0.05). It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2. RANTES and CCR5 may become a marker of gastric cancer metastasis.

Microcalorimetric studies of the synergistic effects of copper-1,10-phenanthroline combined with hyperthermia on a liver hepatoma cell line Bel-7402

The effects of copper-1,10-phenanthroline combined with hyperthermia on human liver hepatoma cell line, Bel-7402 were studied. The effect was evaluated by the mean thermal power of the cells and total energy Q produced during the measurement period (35 h). It was found that the energy produced reduced after the treatment. Condensation of nuclear chromatin and apoptotic bodies can be observed from fluorescence microscope, which showed apoptosis occurs under the action of copper-1,10-phenanthroline combined with hyperthermia. The analysis by flow cytometry showed the proportion of apoptotic cells in the cell population increased. It indicates that the combination of hyperthermia and copper-1,10-phenanthroline has a synergetic effect on Bel-7402.