Requirement of SLD5 for early embryogenesis.

SLD5 forms a GINS complex with PSF1, PSF2 and PSF3, which is essential for the initiation of DNA replication in lower eukaryotes. Although these components are conserved in mammals, their biological function is unclear. We show here that targeted disruption of SLD5 in mice causes a defect in cell proliferation in the inner cell mass, resulting in embryonic lethality at the peri-implantation stage, indicating that SLD5 is essential for embryogenesis. Moreover, this phenotype of SLD5 mutant mice is quite similar compared with that of PSF1 mutant mice. We have previously reported that haploinsufficiency of PSF1 resulted in failure of acute proliferation of bone marrow hematopoietic stem cells (HSCs) during reconstitution of bone marrow ablated by 5-FU treatment. Since SLD5 was highly expressed in bone marrow, we investigated its involvement in bone marrow reconstitution after bone marrow ablation as observed in PSF1 heterozygous mutant mice. However, heterozygous deletion of the SLD5 gene was found not to significantly affect bone marrow reconstitution. On the other hand, abundant SLD5 expression was observed in human cancer cell lines and heterozygous deletion of the gene attenuated tumor progression in a murine model of spontaneous gastric cancer. These indicated that requirement and dependency of SLD5 for cell proliferation is different in different cell types.

PSF3 marks malignant colon cancer and has a role in cancer cell proliferation.

PSF3 (partner of Sld five 3) is a member of the tetrameric complex termed GINS, composed of SLD5, PSF1, PSF2, and PSF3, and well-conserved evolutionarily. Previous studies suggested that some GINS complex members are upregulated in cancer, but PSF3 expression in colon carcinoma has not been investigated. Here, we established a mouse anti-PSF3 antibody, and examined PSF3 expression in human colon carcinoma cell lines and colon carcinoma specimens. We found that PSF3 is expressed in the crypt region in normal colonic mucosa and that many PSF3-positive cells co-expressed Ki-67. This suggests that PSF3-positivity of normal mucosa is associated with cell proliferation. Expression of the PSF3 protein was greater in carcinoma compared with the adjacent normal mucosa, and even stronger in high-grade malignancies, suggesting that it may be associated with colon cancer progression. PSF3 gene knock-down in human colon carcinoma cell lines resulted in growth inhibition characterized by delayed S-phase progression. These results suggest that PSF3 is a potential biomarker for diagnosis of progression in colon cancer and could be a new target for cancer therapy.

PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.

PSF1 (partner of sld five 1) is an evolutionarily conserved DNA replication factor implicated in DNA replication in lower species that is strongly expressed in a wide range of normal stem cell populations and progenitor cell populations. Because stem and progenitor cells possess high proliferative capacity, we hypothesized that PSF1 may play an important role in tumor growth. To begin to investigate PSF1 function in cancer cells, we cloned the mouse PSF1 promoter and generated lung and colon carcinoma cells that stably express a PSF1 promoter-reporter gene. Reporter expression in cells correlated with endogenous PSF1 mRNA expression. In a tumor cell xenograft model, high levels of reporter expression correlated with high proliferative activity, serial transplantation potential, and metastatic capability. Notably, cancer cells expressing reporter levels localized to perivascular regions in tumors and displayed expression signatures related to embryonic stem cells. RNAi-mediated silencing of endogenous PSF1 inhibited cancer cell growth by disrupting DNA synthesis and chromosomal segregation. These findings implicate PSF1 in tumorigenesis and offer initial evidence of its potential as a theranostic target.

Identification and characterization of stem cell-specific transcription of PSF1 in spermatogenesis.

PSF1 is an evolutionarily conserved DNA replication factor, which forms the GINS complex with PSF2, PSF3, and SLD5. The mouse PSF1 homolog has been identified from a stem cell-specific cDNA library. To investigate its transcriptional regulatory mechanisms during differentiation, we studied PSF1 mRNA expression in testis and characterized its promoter. No canonical TATA or CAAT boxes could be found in the PSF1 5'-flanking region, whereas several consensus AML1, GATA, and Sry putative binding sequences are predicted within 5 kb of the putative transcription start site. In addition, binding sites for oncoproteins such as Myb and Ets were also found in the promoter. In testis, various PSF1 gene transcription initiation sites are present and short transcripts encoding two novel isoforms, PSF1b and 1c, were found. However, spermatogonium stem cells specifically express transcripts for PSF1a. These data suggest that PSF1 is tightly regulated at the transcriptional level in stem cells.

Down-regulation of cobalt-induced HIF-1alpha expression correlates with cell proliferation and apoptosis in human gastric carcinoma cells.

Previously we reported that the hypoxia-inducible factor-1alpha (HIF-1alpha) expression correlated with cell proliferation and apoptosis under 500 mM of CoCl2 treatment in a human gastric carcinoma cell line, MKN-1. Herein we report a similar correlation in other cell lines, MKN-45 and TMK-1. The dual-phase expression of HIF-1alpha was highest at 6 and 8 h of treatment in MKN-45 and TMK-1, respectively, while the peak in MKN-1 occurred at 4 h. The cell viability indices showed a similar dual phase to the HIF-1alpha expression, while the apoptotic indices started to increase as the level of the HIF-1alpha expression decreased. In our previous study, the FACS analysis showed a marked G2/M arrest and an increase of the pre-G1 area in MKN-1 after 36 h of treatment, whereas the G2/M arrest was not observed in MKN-45 and TMK-1. The expression of cell cycle and apoptosis-related proteins showed a correlation with the HIF-1alpha expression and the FACS results, which suggested that the level of HIF-1alpha correlated with proliferation and apoptosis in human gastric carcinoma cell lines with a possible cell-type specific pattern.

Apoptotic pathway induced by transduction of RUNX3 in the human gastric carcinoma cell line MKN-1.

The human runt-related transcription factor 3 gene (RUNX3) is considered to be a candidate tumor suppressor gene in gastric carcinoma. However, the role of RUNX3 in the regulation of cell proliferation remains unclear. In the present study, we constructed an adenoviral vector encoding human RUNX3 cDNA under the control of a Tet-responsive promoter (Ad-Tet-FLAG-RUNX3), which regulates the expression of RUNX3 in the presence or absence of doxycycline. A recombinant adenoviral expression vector encoding LacZ (Ad-Tet-LacZ) was used as a negative control. The effect of the transduction of RUNX3 on cell growth was examined using the Tet-On system in a human gastric carcinoma cell line, MKN-1. Exogenous RUNX3 expression was induced successfully by Ad-Tet-FLAG-RUNX3, but not Ad-Tet-LacZ, in the presence of doxycycline in the MKN-1 cells. At 72 h after infection, the proliferative activity in RUNX3-expressing cells was 55% or less of that of the control cells. Flow cytometry revealed that the sub-G(1) peak was increased in cells expressing RUNX3 (34.11%), indicating that the inhibition of cell growth was due to apoptosis, which was confirmed based on Hoechst 33258 staining, the release of cytochrome c from mitochondria into the cytosol, and detection of cleaved caspase-3 by western blotting in MKN-1 cells. Comprehensive analysis using a cDNA microarray showed that RUNX3 upregulated 17 apoptosis-related genes (including FADD, TRAF6, caspase-2, ING1, ING4, Calpain 10, and DNase1) and downregulated 135 apoptosis-related genes (including FLIP, PEA15, TXN2, HSPD1, IKK, and TIAL1) in MKN-1 cells. Pathway analyses to generate functional networks of the genes suggested that promotion of the formation of the death-inducing signaling complex and activation of the mitochondria-mediated pathway were associated with RUNX3-induced apoptosis. In conclusion, our findings suggest that exogenous RUNX3 expression suppressed cell proliferation by inducing apoptosis via the death-receptor mitochondria-mediated pathway in MKN-1 cells.

EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis.

EphB4 receptor and its ligand ephrinB2 play an important role in vascular development during embryogenesis. In blood vessels, ephrinB2 is expressed in arterial endothelial cells (EC) and mesenchymal supporting cells, whereas EphB4 is only expressed in venous ECs. Previously, we reported that OP9 stromal cells, which support the development of both arterial and venous ECs, in which EphB4 was overexpressed, could inhibit ephrinB2-positive (ephrinB2+) EC development in an embryonic tissue organ culture system. Although the EphB4 receptor is expressed in a variety of tumor cells, its exact function in regulating tumor progression has not been clearly shown. Here we found that overexpression of EphB4 in B16 melanoma cells suppressed tumor growth in a s.c. transplantation tumor model. Histologic examination of these tumors revealed that EphB4 overexpression in B16 cells selectively suppressed arterial ephrinB2+ EC development. By coculturing ephrinB2-expressing SV40-transformed mouse ECs (SVEC) with EphB4-overexpressing B16 cells, we found that EphB4 induced the apoptosis of SVECs. However, ephrinB2 did not induce the apoptosis of EphB4-overexpressing B16 cells. Based on results from these experiments, we concluded that EphB4 overexpression in B16 tumor cells suppresses the survival of arterial ECs in tumors by a reverse signaling via ephrinB2.

Runt-related transcription factor 3 expression in human oral squamous cell carcinomas; implication for tumor progression and prognosis.

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor factor of gastric cancer and appears to be an important component of the transforming growth factor-beta (TGF-beta)-induced tumor suppression pathway. This study aimed to analyze the expression of the RUNX3 protein in human oral normal epithelia, dysplasia and squamous cell carcinomas (SCCs), comparing it with clinicopathological profiles. Western blot analysis revealed the RUNX3 protein as a single band at 44kDa in oral non-neoplastic mucosa and SCC. The expression of RUNX3 protein was also examined in 10 normal epithelia, 51 dysplasias and 108 oral SCCs. The labeling indices (LIs) of RUNX3, Ki-67, P21, P27 and the apoptotic index (AI) were evaluated using immunohistochemistry and the TUNEL method. The LI of RUNX3 was 7.7+/-1.6 in the normal epithelia, 20.8+/-2.7 in the dysplasias and 9.0+/-1.3 in the SCCs. The LI of RUNX3 was significantly highest in the dysplasias, followed by the SCCs (p<0.05) and normal epithelia (p<0.05). The RUNX3 LI correlated with the histological differentiation of SCCs, being the highest in the well differentiated SCCs (p<0.01). In addition, RUNX3 expression was significantly related to the lower Ki-67 LI, but not to LI of P21 and P27, and AI in the SCCs. The survival rate was significantly lower in the patients with lower RUNX3 expression (<5%) than in those with higher expression (5%) (p<0.05). These results indicate that the expression of RUNX3 is correlated with histological differentiation, and inversely with cellular proliferation of the oral SCCs, and might be a new prognostic marker in the patients with oral SCC.

CoCl2-induced HIF-1alpha expression correlates with proliferation and apoptosis in MKN-1 cells: a possible role for the PI3K/Akt pathway.

The exact mechanism behind the effect of hypoxia-inducible factor-1alpha (HIF-1alpha) on the proliferation and/or apoptosis of carcinoma cells is still a matter of debate. We treated a human gastric carcinoma cell line, MKN-1 (mutant P53), with 500 microM CoCl(2). A dual-phase pattern of HIF-1alpha expression with an increase until 4 h followed by a decrease until 36 h was observed. Immunocytochemistry showed that nuclear translocation was maximal at 4 h of treatment, while trypan blue staining showed a dual-phase pattern. Instead of G1/S arrest, FACS showed an increase in the pre-G1 fraction and G(2)/M arrest that correlated with Cyclin-B1, SKP-2 and P27 expression. Starting at 6 h, the apoptotic index increased in a time-dependent manner, in correlation with the expression of HIF-1alpha, Bcl-2, Bcl-xL, Bax and cleaved-Caspase-9. Phosphorylation of Akt was inhibited by CoCl(2) treatment and LY294002 treatment inhibited HIF-1alpha expression in a dose-dependent manner. These results suggested that the alteration of CoCl(2)-induced HIF-1alpha expression correlated with proliferation and apoptosis in MKN-1 cells. A possible role for the PI3K/Akt pathway was indicated in this model of hypoxia.

Expression of RUNX3 protein in human lung adenocarcinoma: implications for tumor progression and prognosis.

Runt-related transcription factor 3 belongs to the runt domain family of transcription factors that play a pivotal role during normal tissue development and tumorigenesis in several organs. We directed our attention to the expression of RUNX3 protein in human lung AC and non-neoplastic lung tissues, comparing the results with clinicopathological profiles. We evaluated the expression of RUNX3 protein in 17 pairs of lung AC and non-neoplastic lung tissue. Furthermore, 98 lung AC were studied to examine the frequency of RUNX3-positive cells. Western blot analysis showed a single band at 45 kDa in all 17 AC and non-neoplastic tissues. Immunohistochemistry revealed immunoreactivity in alveolar type II pneumocytes or Clara cells. RUNX3 was expressed more frequently in the carcinomas with a BAC component than in those without (P < 0.01). Lower RUNX3 levels were associated with poorly differentiated types (P = 0.049). The five-year survival rate was significantly higher in the 50 patients with higher levels of RUNX3 expression than in the 48 patients with lower levels (P = 0.027). The expression of RUNX3 protein in lung AC might play a pivotal role in tumor progression and patients' survival.