Cell growth regulation through apoptosis by activin in human gastric cancer SNU-16 cell lines.

Activin has a wide variety of biological functions, including the regulation of cell proliferation and inhibition of tumor cells. We have studied whether activin regulates apoptosis by investigating the effects of activin A on cell proliferation, cell cycle, apoptosis, apoptosis-related gene expression, and caspase activity in SNU-16 cells. Activin A significantly inhibited DNA synthesis and growth suppression in a time-dependent manner in SNU-16 cells. Apoptosis fraction was increased at cell cycle with an accompanying DNA fragmentation. Activin A resulted in a significant time-dependent decrease in Bcl-2 mRNA levels and increase in caspase-3 mRNA levels in SNU-16 cells. No significant difference was observed in Bax mRNA levels. Exposure of cells to activin A induced caspase-3, -8 and -9 activation in SNU-16 cells. Furthermore, co-treatment of activin with the pan-caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Z-DEVE-FMK, caspase-8 inhibitor Z-IETD-FMK, and caspase-9-inhibitor Z-LEHD-FMK blocked apoptosis of SNU-16 cells. Taken together, our results revealed that activin inhibits the growth of SNU-16 cells by inducing apoptosis through caspase activation.

Selective inhibition of cell growth by activin in SNU-16 cells.

AIM: To investigate whether activin regulates the cell proliferation of human gastric cancer cell line SNU-16 through the mRNA changes in activin receptors, Smads and p21(CIP1/WAF1). METHODS: The human gastric cancer cell lines were cultured, RNAs were purified, and RT-PCRs were carried out with specifically designed primer for each gene. Among them, the two cell lines SNU-5 and SNU-16 were cultured with activin A for 24, 48 and 72 h. The cell proliferation was measured by MTT assay. For SNU-16, changes in ActRIA, ActRIB, ActRIIA, ActRIIB, Smad2, Smad4, Smad7, and p21(CIP1/WAF1) mRNAs were detected with RT-PCR after the cells were cultured with activin A for 24, 48 and 72 h. RESULTS: The proliferation of SNU-16 cells was down regulated by activin A whereas other cells showed no change. Basal level of inhibin/activin subunits, activin receptors, Smads, and p21(CIP1/WAF1) except for activin betaB mRNAs was observed to have differential expression patterns in the human gastric cancer cell lines, AGS, KATO III, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-638, SNU-668, and SNU-719. Interestingly, significantly higher expressions of ActR IIA and IIB mRNAs were observed in SNU-16 cells when compared to other cells. After activin treatment, ActR IA, IB, and IIA mRNA levels were decreased whereas ActR IIB mRNA level increased in SNU-16 cells. Smad4 mRNA increased for up to 48 h whereas Smad7 mRNA increased sharply at 24 h and returned to the initial level at 48 h in SNU-16 cells. In addition, expression of the p21(CIP1/WAF1), the mitotic inhibitor, peaked at 72 h after activin treatment in SNU-16 cells. CONCLUSION: Our results suggest that inhibition of cell growth by activin is regulated by the negative feedback effect of Smad7 on the activin signaling pathway, and is mediated through p21(CIP1/WAF1) activation in SNU-16 cells.

Involvement of CLOCK:BMAL1 heterodimer in serum-responsive mPer1 induction.

A rapid induction of mouse period1 (mPer1) gene expression is supposed to be critical in the clock gene regulation, especially in the phase resetting of the clock, but its molecular mechanism is poorly understood. Based on the previous finding that the process does not involve de novo synthesis of proteins, we postulated the involvement of CLOCK:BMAL1 heterodimer, a positive regulator of circadian oscillator, in the rapid induction of mPer1 transcription. To test this hypothesis, we utilized CLOCKdelta19, a dominant-negative mutant, to suppress the function of CLOCK:BMAL1 in vitro. Serum-evoked rapid increases of mPer1 mRNA expression and promoter activity were significantly blunted when CLOCK:BMAL1 function was interfered with. Furthermore, DNA binding activity of CLOCK:BMAL1 heterodimer to five E-boxes of mPer1 promoter markedly increased shortly after serum shock. Taken together, these results suggest that CLOCK:BMAL1 heterodimer is not only a core component of negative feedback loop driving circadian oscillator, but also involved in the rapid induction of mPer1during phase resetting of the clock.

Expression of epithin in mouse preimplantation development: its functional role in compaction.

The preimplantation development of mammalian embryo after fertilization encompasses a series of events including cleavage, compaction, and differentiation into blastocyst. These events are likely to be associated with substantial changes in embryonic gene expression. In the present study, we explored the expression patterns and function of epithin, a mouse type II transmembrane serine protease, during preimplantation embryo development. RT-PCR analysis showed that epithin mRNAs were detectable during the cleavage stages from a 1-cell zygote to the blastocyst. Immunocytochemical studies revealed that epithin protein was expressed at blastomere contacts of the compacted 8-cell and later embryonic stages. Epithin colocalized with E-cadherin at the membrane contacts of the compacted morula-stage embryo as revealed by double-staining immunocytochemistry and confocal microscopy, respectively. Post-transcriptional epithin gene silencing by RNA interference (RNAi) resulted in the blockade of 8-cell in vitro-stage embryo compaction and subsequent embryonic deaths after several rounds of cell division. These results strongly suggest that epithin plays an important role in the compaction processes that elicit the signal for the differentiation into trophectoderm and inner cell mass.

Suppression of Nek2A in mouse early embryos confirms its requirement for chromosome segregation.

Nek2, a mammalian structural homologue of Aspergillus protein kinase NIMA, is predominantly known as a centrosomal kinase that controls centriole-centriole linkage during the cell cycle. However, its dynamic subcellular localization during mitosis suggested that Nek2 might be involved in diverse cell cycle events in addition to the centrosomal cycle. In order to determine the importance of Nek2 during mammalian development, we investigated the expression and function of Nek2 in mouse early embryos. Our results show that both Nek2A and Nek2B were expressed throughout early embryogenesis. Unlike cultured human cells, however, embryonic Nek2A appeared not to be destroyed upon entry into mitosis, suggesting that the Nek2A protein level is controlled in a unique manner during mouse early embryogenesis. Suppression of Nek2 expression by RNAi resulted in developmental defects at the second mitosis. Many of the blastomeres in Nek2-suppressed embryos showed abnormality in nuclear morphology, including dumbbell-like nuclei, nuclear bridges and micronuclei. These results indicate the importance of Nek2 for proper chromosome segregation in embryonic mitoses.

Expression pattern of HSP25 in mouse preimplantation embryo: heat shock responses during oocyte maturation.

Heat shock proteins (HSPs) are known to play an important role not only in various stress conditions such as exposure to heat shock, but also in normal development and/or differentiation. The role of small heat shock proteins such as HSP25 in early embryo development remains largely unknown. In the present study, we examined temporal and spatial expression patterns of HSP25 during mouse preimplantation embryo development. Reverse transcription-polymerase chain reaction (RT-PCR) showed that hsp25 mRNA was detected in unfertilized eggs. Hsp25 mRNA was induced by zygotic gene activation at 2-cell stage, decreased slightly at 4-cell, and re-increased at morula, with the highest level at blastocyst stage. Interestingly, another form of hsp25 variant of which 156 bp (52 a.a.) was truncated within the exon1 region was observed in all stages of preimplantation embryos. We also investigated the sub-cellular localization of HSP25 by fluorescence immunocytochemistry. HSP25 was detected in the cytoplasm under normal developmental condition. While acute heat shock (at 43 degrees C for 30 min) caused no significant changes in the sub-cellular localization of HSP25 in the developing mouse embryos, chronic heat shock (at 43 degrees C for 3 hr) resulted in a denser immunostaining of HSP25 in the nucleus than in the cytoplasm, indicating a nuclear translocation of HSP25 by heat shock. As hsp25 mRNA was detected in the unfertilized egg as a maternal transcript, we examined the expression of hsp25 mRNA with RT-PCR during oocyte maturation under normal and heat shock conditions. Hsp25 mRNA was detected at GV (germinal vesicle)-, GVBD (germinal vesicle breakdown)-, and MII (metaphase II)-oocytes. The expression of hsp25 mRNA was increased markedly by both acute (for 30 min and 1 hr) and chronic (for 4 hr) heat shock, but returned to the basal level during recovery from heat shock in a time-dependent manner, suggesting a thermo-protective role of HSP25. In contrast to preimplantation embryos, HSP25 was detected both in the cytoplasm and the nucleus except for the nucleolus, and the cellular localization was not altered by heat shock. Finally, we investigated the effect of heat shock on oocyte maturation. When GV-oocytes were exposed to acute heat shock (at 43 degrees C for 15 min to 1 hr), they underwent the GVBD and the PB (polar body) emission successfully. However, under more stringent heat shock conditions (at 43 degrees C for 2-4 hr), most oocytes were arrested at the GV-stage, and the first PB was not developed, indicating that chronic heat shock might be inhibitory to the mouse oocyte maturation. Taken together, these findings suggest that HSP25 is important for mouse preimplantation embryo development and oocyte maturation.

Identification of estrogen-regulated genes in the mouse uterus using a delayed-implantation model.

Gonadal steroid hormones are known to modulate the implantation of the blastocyst, but how the controlling genetics are regulated remains largely unknown. Using a delayed-implantation model, we examined estrogen-regulated genes (ERGs) in the mouse uterus using the differential-display reverse transcription-polymerase chain reaction (DD RT-PCR). Pregnant mice were ovariectomized and injected daily with progesterone (P, 1 mg/mouse), followed by a single injection of estrogen (E, 200 ng/mouse); 24 or 48 hr later, total RNA was extracted from the uterus. Reverse Northern analysis verified the expression patterns of 36 clones out of thousands of RNA species. Only five clones had mRNA levels that were modified, whereas other mRNAs were unchanged or not detectable. Sequence analysis of these, using the Basic Local Alignment Search Tool (BLAST) service, revealed that four of these clones were novel; one clone, designated ERG10, was found to be the mouse homologue of that deleted in oral cancer DOC-1. DOC-1 mRNA was detected all tissues examined, but only in the uterus and cervix was markedly increased 12 hr after E administration, it returned to basal level by 48 hr. One of the novel genes, designated ERG8, had three different forms of mRNAs and was expressed ubiquitously in all examined tissues. In the uterus, the mRNA level of ERG8 also increased 12 hr after E administration. These results suggest that during the implantation process, E differentially regulates several genes depending on cell type. Uterine-specific induction of newly found genes, such as ERG8 and 10, by E appears to be important for the early implantation process.