• Molecules and Cells
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• v.41 no.7
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• pp.684-694
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• 2018

Upregulation of human telomerase reverse transcriptase (hTERT) expression is an important factor in the cellular survival and cancer. Although growing evidence suggests that hTERT inhibits cellular apoptosis by telomere-independent functions, the mechanisms involved are not fully understood. Here, we show that hTERT contains a BH3-like motif, a short peptide sequence found in BCL-2 family proteins, and interacts with anti-apoptotic BCL-2 family proteins MCL-1 and BCL-xL, suggesting a functional link between hTERT and the mitochondrial pathway of apoptosis. Additionally, we propose that hTERT can be categorized into the atypical BH3-only proteins that promote cellular survival, possibly due to the non-canonical interaction between hTERT and antiapoptotic proteins. Although the detailed mechanisms underlying the hTERT BH3-like motif functions and interactions between hTERT and BCL-2 family proteins have not been elucidated, this work proposes a possible connection between hTERT and BCL-2 family members and reconsiders the role of the BH3-like motif as an interaction motif.

• Development and Reproduction
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• v.15 no.2
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• pp.113-120
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• 2011

BCL-2 family essential proteins to play a pivotal role to perform in apoptosis signaling pathways and essential proteins for the regulation of cell death. BCL2L10 protein is a member of BCL-2 family and it regulates both anti-apoptotic and pro-apoptotic function of specific tissue or cell line. BCL2L10 of function and expression is not reported in ovary cell lines. In this study we reported that BCL2L10 were significant expression of KGN cell line. Ectopic expression of BCL2L10 induced cell death, and its cells killing effect was blocked by pan-caspase inhibitor of the Z-VAD-fmk. Ectopic expression of BCL2L10 protein led to the activation of caspase 9 and caspase 3, suggesting apoptotic cell death, and confocal microscopic analyses showed that BCL2L10 was partially localized in mitochondria. Thus, we provide a novel function of BCL2L10 in KGN cells, which was involved in the intrinsic cell death pathway.

• Molecules and Cells
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• v.37 no.3
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• pp.264-269
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• 2014

The molecular interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins plays an essential role in the transcription-independent apoptotic pathway of p53. In this study, we investigated the binding of p53 DNA-binding domain (p53DBD) with the anti-apoptotic Bcl-2 family proteins, Bcl-w, Mcl-1, and Bcl-2, using GST pull-down assay and NMR spectroscopy. The GST pull-down assays and NMR experiments demonstrated the direct binding of the p53DBD with Bcl-w, Mcl-1, and Bcl-2. Further, NMR chemical shift perturbation data showed that Bcl-w and Mcl-1 bind to the positively charged DNA-binding surface of p53DBD. Noticeably, the refined structural models of the complexes between p53DBD and Bcl-w, Mcl-1, and Bcl-2 showed that the binding mode of p53DBD is highly conserved among the anti-apoptotic Bcl-2 family proteins. Furthermore, the chemical shift perturbations on Bcl-w, Mcl-1, and Bcl-2 induced by p53DBD binding occurred not only at the p53DBD-binding acidic region but also at the BH3 peptide-binding pocket, which suggests an allosteric conformational change similar to that observed in Bcl-$X_L$. Taken altogether, our results revealed a structural basis for a conserved binding mechanism between p53DBD and the anti-apoptotic Bcl-2 family proteins, which shed light on to the molecular understanding of the transcription-independent apoptosis pathway of p53.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.27-33
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• 1999

In the mammalian ovary, follicular development and atresia continuously occur during the reproductive cycles. Follicular atresia occurs through granulosa cell apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2 and bcl-xLong are known as inhibitors of apoptosis, whereas bax and bcl-xShort are known as inducer of apoptosis. We thought that bcl-2 protein is associated with follicular development and atresia. But it is not known that the distribution of cells containing bcl-2 protein during follicular development and atresia. Therefore, to examine the distribution of cells with bcl-2 protein during ovarian follicular development and atresia, the immunohistochemistry was used in the rat ovary. Bcl-2 immunoreactivity was localized in the interstitial cells, theca externa cells and granulosa cells around of antrum. All positive signals were observed in the cytoplasm of these cells. Positive signals were strongly observed in the interstitial and theca externa cells of growing antral follicles. While, positive signals were weakly observed in these cells from atretic antral follicles. Positive signals were very weakly observed in the granulosa cells of growing and atretic antral follicles. According to these data, we suggested that bcl-2 proteins which were strongly expressed in the interstitial cells and theca externa cells of growing antral follicles inhibit follicular atresia. And we purposed that bcl-2 proteins regulated follicular development and atresia through the action of bcl-2 gene family.

• Journal of Oral Medicine and Pain
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• v.30 no.3
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• pp.301-317
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• 2005

The number of patients with tongue carcinoma is increasing rapidly among young individuals in many parts of the world. Oral carcinoma progresses from hyperplastic lesion through dysplasia to invasive carcinoma and the concept of "field cancerization" with molecular alteration has been suggested for oral cavity carcinogenesis. Significant improvement in treatment and prognosis will depend on more detailed understanding of the multi-step process leading to cancer development. To induce tongue carcinoma in rat by 4-NQO, each drinking water was made to 10 ppm, 25 ppm, 50 ppm and control (only D.W. without 4-NQO). Specimens were classified into 4 groups such as control, I (mild & moderate dysplasia), II (severe dysplasia and carcinoma in situ), III (carcinoma). The mRNA expressions of Bcl-2 family were evaluated by RT-PCR technique. For anti-apoptotic Bcl-2 family, mRNA expression of Bcl-w was down-regulated in all stages of tongue carcinogenesis model. However, mRNA expression of Bcl-2 was up-regulated. For pro-apoptotic Bcl-2 family, all members were down-regulated in all stages of tongue carcinogenesis model except for Bad mRNA in group III. In terms of BH3 only protein, mRNA expressions of Bok and Mcl-1 were down regulated in all stages of specimen, but Bmf in group II and BBC3 in group III were up-regulated. Our current findings demonstrated the involvements of mRNA expression of Bcl-2 family in multi-step tongue carcinogensis. This highlights the necessity for continued efforts to discover suitable biomakers (Bcl-2 family) for early diagnosis of the disease, and to understand its pathogenesis as a first step in improving methods of treatment. The discovery of these potential biomarkers and molecular targets for cancer diagnostics and therapeutics has the potential to significantly change the clinical approach and outcome of the disease.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2002.07a
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• pp.113-113
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• 2002

Phylogenetically conserved Bcl-2 family proteins play a pivotal role in the regulation of apoptosis from virus to human. Members of the Bcl-2 family consist of antiapoptotic proteins such as Bcl-2, Bcl-xL, and Bcl-w, and proapoptotic proteins such as BAD, Bax, BOD, and Bok. It has been proposed that anti- and proapoptotic Bcl-2 proteins regulate cell death by binding to each other and forming heterodimers. A delicate balance between anti- and proapoptotic Bcl-2 family members exists in each cell and the relative concentration of these two groups of proteins determines whether the cell survives or undergoes apoptosis. Mcl-1 (Myeloid cell :leukemia-1) is a member of the Bcl-2 family proteins and was originally cloned as a differentiation-induced early gene that was activated in the human myeloblastic leukemia cell line, ML-1 . Mcl-1 is expressed in a wide variety of tissues and cells including neoplastic ones. We recently identified a short splicing variant of Mcl-1 short (Mcl-IS) and designated the known Mcl-1 as Mcl-1 long (Mcl-lL). Mcl-lL protein exhibits antiapoptotic activity and possesses the BH (Bcl-2 homology) 1, BH2, BH3, and transmembrane (TM) domains found in related Bcl-2 proteins. In contrast, Mcl-1 S is a BH3 domain-only proapoptotic protein that heterodimerizes with Mcl-lL. Although both Mc1-lL and Mcl-lS proteins contain BH domains fecund in other Bcl-2 family proteins, they are distinguished by their unusually long N-terminal sequences containing PEST (proline, glutamic acid, serine, and threonine) motifs, four pairs of arginine residues, and alanine- and glycine-rich regions. In addition, the expression pattern of Mcl-1 protein is different from that of Bcl-2 suggesting a unique role (or Mcl-1 in apoptosis regulation. Tankyrasel (TRF1-interacting, ankyrin-related ADP-related polymerasel) was originally isolated based on its binding to TRF 1 (telomeric repeat binding factor-1) and contains the sterile alpha motif (SAM) module, 24 ankyrin (ANK) repeats, and the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP). Previous studies showed that tankyrasel promotes telomere elongation in human cells presumably by inhibiting TRFI though its poly(ADP-ribosyl)action by tankyrasel . In addition, tankyrasel poly(ADP-ribosyl)ates Insulin-responsive amino peptidase (IRAP), a resident protein of GLUT4 vesicles, and insulin stimulates the PARP activity of tankyrase1 through its phosphorylation by mitogen-activated protein kinase (MAPK). ADP-ribosylation is a posttranslational modification that usually results in a loss of protein activity presumably by enhancing protein turnover. However, little information is available regarding the physiological function(s) of tankyrase1 other than as a PARP enzyme. In the present study, we found tankyrasel as a specific-binding protein of Mcl-1 Overexpression of tankyrasel led to the inhibition of both the apoptotic activity of Mel-lS and the survival action of Mcl-lL in mammalian cells. Unlike other known tankyrasel-interacting proteins, tankyrasel did not poly(ADP-ribosyl)ate either of the Mcl-1 proteins despite its ability to decrease Mcl-1 proteins expression following coexpression. Therefore, this study provides a novel mechanism to regulate Mcl-1-modulated apoptosis in which tankyrasel downregulates the expression of Mcl-1 proteins without the involvement of its ADP-ribosylation activity.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.167-172
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• 2012

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 ($14.7{\pm}3.0$ vs $30.3{\pm}4.8%$, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

• Journal of Gastric Cancer
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• v.3 no.2
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• pp.84-87
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• 2003

Purpose: Evidence exists that dysregulation of Bcl-2 family members is involved in the pathogenesis of cancer development. The aim of this study was to explore whether the somatic mutation of proapoptotic Bcl-2 member genes, one of the mechanisms that prolong the survival of cancer cells, is involved in gastric carcinogenesis. Materials and Methods: In the current study, to detect somatic mutations of the DNA sequences encoding the Bcl-2 homology 3 (BH3) domain of the human BAD, BIM, BIK, and Bcl-G genes in 60 advanced gastric adenocarcinomas, we used the polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP), and DNA sequencing. Results: The SSCP analysis revealed no mutations in the coding regions of the BH3 domain in the cancers. Conclusion: The data presented here indicate that proapoptotic Bcl-2 member genes, BAD, BIM, BIK, and Bcl-G, may not be mutated in human gastric carcinomas and suggest that these genes might be altered by mechanisms other mechanisms somatic mutation.

• Tuberculosis and Respiratory Diseases
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• v.56 no.5
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• pp.450-464
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• 2004

Background : The FHIT (fragile histidine triad) gene is a frequent target of deletions associated with abnormal RNA and protein expression in lung cancer. Previous studies have shown FHIT gene transfer into lung cancer cell line lacking FHIT protein expression resulted in inhibition of tumor cell growth attributable to the induction of apoptosis and reversion of tumorigenecity. However, the mechanism of the tumor suppressor activity of the FHIT gene and the cellular pathways associated with its function are not completely understood. Methods : To gain insight into the biological function of FHIT, we compared the NCI-H358 cell line with its stable FHIT transfectants after treatment with cisplatin or paclitaxel. We investigated the effects of FHIT gene expression on cell proliferation, apoptosis, and activation of caspase system and Bcl-2 family. The induction of apoptosis was evaluated by using DAPI staining and flow cytometry. Activation of caspases and Bcl-2 members was evaluated by Western blot analysis. Results : A significantly increased cell death was observed in FHIT transfectants after cisplatin or paclitaxel treatment and this was attributable to the induction of apoptosis. Remarkable changes in caspases and Bcl-2 family were observed in the transfected cells as compared with the control cells after treatment with paclitaxel. Activation of caspase-3 and caspase-7 was markedly increased in cells expressing FHIT. Expression level of Bcl-2 and Bcl-xL protein was significantly decreased and that of Bax and Bad protein was significantly increased in the transfected cells. Conclusion : FHIT gene delivery into lung cancer cells results in enhanced apoptosis induced by treatment with cisplatin or paclitaxel. The data suggest that apoptosis in FHIT-expressing cells could be related to activation of caspase pathway and Bcl-2 family.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.731-745
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• 2005

Cyclosproin A(CsA)는 세포 이식거부방지를 위한 면역 억제제 및 자가 면역질환 치료제로 널리 사용되어 왔다. CsA는 매양된 사람 치은섬유아세포를 증식시킴이 알려져 있지만 CsA에 의한 세포증식기전에 대한 세포사멸기전 및 Bcl-2의 역할은 연구되어 있지 않다. 이번 연구는 사람 섬유아세포에서 CsA에 의한 세포증삭기전에 세포고사기전 및 Bcl-2 family가 관여하는지 밝히는 데에 목적이 있다. 세포 생장력은 MTT 방법으로 측정하였다. Bcl-2 family와 Fas 발현 정도는 RT-PCR 방법이나 western blot으로 확인하였다. Caspase-3 및 -9의 활성은 ELISER reader로, reactive oxygen species(ROS)는 fluorescence spectrometer에 의해 측정되었다. 미토콘드리아에서 세포질로 분비된 cytochrome c는 Western blot으로 조사하였다. CsA는 $0.1{\sim}10\;{\mu}M$에서 사람 섬유아세포의 생존률을 시간과 농도 의존적으로 증가시켰으며, 50 ${\mu}M$ CsA에서는 오히려 세포가 죽였다. 또한, CsA 처리로 미토콘드리아에서 세포질로 유리되는 cytochrome c 양과 VDAC 1 및 3 발현량이 감소되었고, caspase-9과 caspase-3의 활성도도 감소되었다. 한편, CsA 처리한 섬유아세포에서 death receptor 구성요소인 Fas 발현이 감소되었다. Bcl-2 family에 대한 RT-PCR, western blot 분석결과, 세포고사를 억제하는 Bel-2 발현은 증가되었으나 세포고사를 자극하는 Bax와 Bid의 발현은 감소되었다. 이러한 결과들은 사람 섬유아세포에서 CsA유도 세포증식에 Bcl-2 family와 ROS가 매개하는 미토콘드리아 의존 및 death receptor 의존 세포고사기전이 관여함을 시사하였다.