Differential interaction of PRMT1 with RGG-boxes of the FET family proteins EWS and TAF15.

The FET sub-family (FUS/TLS, EWS, TAF15) of RNA-binding proteins have remarkably similar overall structure but diverse biological and pathological roles. The molecular basis for FET protein specialization is largely unknown. Gly-Arg-Rich regions (RGG-boxes) within FET proteins are targets for methylation by Protein-Arginine-Methyl-Transferase-1 (PRMT1) and substrate capture is thought to involve electrostatic attraction between positively charged polyRGG substrates and negatively charged surface channels of PRMT1. Unlike FUS and EWS, a high proportion of TAF15 RGG-boxes are embedded within neutrally charged YGGDR(S/G)G repeats, suggesting that they might not bind well to PRMT1. This notion runs contrary however to a report that YGGDR(S/G)G repeats are methylated by PRMT1. Using peptide-based polyRGG substrates and a novel 2-hybrid binding assay, we find that the Asp residue in YGGDR(S/G)G repeats confers poor binding to PRMT1. Our results therefore indicate that YGGDR(S/G)G repeats may contribute to TAF15 specialization by enabling differential interactions with PRMT1 and reduced overall levels of TAF15 methylation compared with other FET proteins. By analogy with molecular recognition of other disordered polyvalent ligands by globular protein partners, we also propose a dynamic polyelectrostatic model for substrate capture by PRMT1.

Mid-term results of the BIOLOX delta ceramic-on-ceramic total hip arthroplasty.

AIMS: We conducted a prospective study of a delta ceramic total hip arthroplasty (THA) to determine the rate of ceramic fracture, to characterise post-operative noise, and to evaluate the mid-term results and survivorship. PATIENTS AND METHODS: Between March 2009 and March 2011, 274 patients (310 hips) underwent cementless THA using a delta ceramic femoral head and liner. At each follow-up, clinical and radiological outcomes were recorded. A Kaplan-Meier analysis was undertaken to estimate survival. RESULTS: Four patients (four hips) died and 18 patients (20 hips) were lost to follow-up within five years. The remaining 252 patients (286 hips) were followed for a mean of 66.5 months (60 to 84). There were 144 men (166 hips) and 108 women (120 hips) with a mean age of 49.7 years (16 to 83) at surgery. The mean pre-operative Harris Hip Score of 47.1 points improved to 93.8 points at final follow-up. Six patients reported squeaking in seven hips; however, none were audible. Radiolucent lines involving Gruen zones one and/or seven were seen in 52 hips (18.2%). No hip had detectable wear, focal osteolysis or signs of loosening. One hip was revised because of fracture of the ceramic liner, which occurred due to an undetected malseating of the ceramic liner at the time of surgery. One hip was revised for a periprosthetic fracture of the femur, and one hip was treated for periprosthetic joint infection. The six-year survivorship with re-operation for any reason as the endpoint was 99.0% (95% confidence interval 97.8% to 100%). DISCUSSION: The rate of delta ceramic fracture was 0.3% (one of 286). While ceramic head fracture was dominant in previous ceramic-on-ceramic THA, fracture of the delta ceramic liner due to malseating is a concern. Cite this article: Bone Joint J 2017;99-B:741-8.

RGG boxes within the TET/FET family of RNA-binding proteins are functionally distinct.

The multi-functional TET (TAF15/EWS/TLS) or FET (FUS/EWS/TLS) protein family of higher organisms harbor a transcriptional-activation domain (EAD) and an RNA-binding domain (RBD). The transcriptional activation function is, however, only revealed in oncogenic TET-fusion proteins because in native TET proteins it is auto-repressed by RGG-boxes within the TET RBD. Auto-repression is suggested to involve direct cation-pi interactions between multiple Arg residues within RGG boxes and EAD aromatics. Via analysis of TET transcriptional activity in different organisms, we report herein that repression is not autonomous but instead requires additional trans-acting factors. This finding is not supportive of a proposed model whereby repression occurs via a simple intramolecular EAD/RGG-box interaction. We also show that RGG-boxes present within reiterated YGGDRGG repeats that are unique to TAF15, are defective for repression due to the conserved Asp residue. Thus, RGG boxes within TET proteins can be functionally distinguished. While our results show that YGGDRGG repeats are not involved in TAF15 auto-repression, their remarkable number and conservation strongly suggest that they may confer specialized properties to TAF15 and thus contribute to functional differentiation within the TET/FET protein family.

Placental deposition of C-reactive protein is a common feature of human pregnancy.

This study examined the occurrence of placental C-reactive protein (CRP) in normal pregnancy with term delivery, spontaneous preterm delivery (sPTD), preeclampsia, and miscarriage. CRP immunoreactivity was detected in the syncytiotrophoblast. The immunopositive rate was significantly higher in sPTD than preeclampsia. The CRP immunopositive rate was also higher in acute chorioamnionitis than those without and showed a good correlation with the maternal serum CRP concentration. CRP mRNA expression was not detected in human and mouse placentas or choriocarcinoma cells. CRP may play a role in the pathological and physiological states of pregnancy.

Extensive curettage using a high-speed burr versus dehydrated alcohol instillation for the treatment of enchondroma of the hand.

The purpose of this retrospective study was to compare the clinical and radiological outcomes of patients treated with different adjuvant methods after curettage for enchondromas of the hand. Sixty-two patients with enchondroma were treated with high-speed burring (29 patients) or alcohol instillation (33 patients) after curettage. The mean follow-up was 40.8 months. No significant differences in the visual analogue scale, Disabilities of the Arm, Shoulder, and Hand scores, total range of active motion, grip strength, and complete healing time were observed between the groups. The distribution of the results of the formula by Wilhelm and Feldmeier were not significantly different between the groups. No surgery-related complications, postoperative pathological fractures, or recurrence was found in either group. For the treatment of enchondroma in the metacarpal and proximal phalanx, alcohol instillation immediately after curettage was as effective as extensive curettage using a high-speed burr.

Endoscopic treatment of gastric band prolapse.

Complications of laparoscopic adjustable gastric banding (LAGB) are well documented including migration, erosion, prolapse, infection, pouch dilatation, and gastric perforation. Band prolapse within the first 5 years after LAGB is observed in about 5% of cases, requiring an operative procedure. Here we report our experience of endoscopic treatment of band prolapses. From December 2007 to December 2013, 1,347 consecutive patients (202 male, 1,145 female) underwent LAGB; 47 patients had band prolapses and 7 were treated by endoscopy. All patients were women (median age, 34 years). The mean preoperative body mass index was 38.3 ± 2.9 kg/m2. The mean duration to band prolapse after LAGB was 10.6 ± 5.6 months. The mean duration of endoscopy was 12 ± 3 min. One patient had recurrence of the prolapse 3 months after the first endoscopy and was treated by endoscopy again. There was no operative procedure required and no mortality. Endoscopic treatment of band prolapses is effective without the need for an operative procedure.

Epigenetic modification of Nrf2 in 5-fluorouracil-resistant colon cancer cells: involvement of TET-dependent DNA demethylation.

5-Fluorouracil (5-FU) is a widely used anticancer drug for the treatment of colorectal cancer (CRC). However, resistance to 5-FU often prevents the success of chemotherapy. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional regulator and a possible target to overcome 5-FU resistance. The present study examined epigenetic changes associated with Nrf2 induction in a human CRC cell line (SNUC5) resistant to 5-FU (SNUC5/5-FUR). Nrf2 expression, nuclear translocation, and binding to promoter were higher in SNUC5/5-FUR cells than in SNUC5 cells. The activated Nrf2 in SNUC5/5-FUR cells led to an increase in the protein expression and activity of heme oxygenase-1 (HO-1), an Nrf2-regulated gene. SNUC5/5-FUR cells produced a larger amount of reactive oxygen species (ROS) than SNUC5 cells. The siRNA- or shRNA-mediated knockdown of Nrf2 or HO-1 significantly suppressed cancer cell viability and tumor growth in vitro and in vivo, resulting in enhanced 5-FU sensitivity. Methylation-specific (MS) or real-time quantitative MS-PCR data showed hypomethylation of the Nrf2 promoter CpG islands in SNUC5/5-FUR cells compared with SNUC5 cells. Expression of the DNA demethylase ten-eleven translocation (TET) was upregulated in SNUC5/5-FUR cells. ROS generated by 5-FU upregulated TET1 expression and function, whereas antioxidant had the opposite effect. These results suggested that the mechanism underlying the acquisition of 5-FU resistance in CRC involves the upregulation of Nrf2 and HO-1 expression via epigenetic modifications of DNA demethylation.

Gracilaria bursa-pastoris (Gmelin) Silva extract attenuates ultraviolet B radiation-induced oxidative stress in human keratinocytes.

The purpose of this study was to assess the protective effects of an ethanol extract derived from the red alga Gracilaria bursa-pastoris (Gmelin) Silva (GBE) on ultraviolet B (UVB)-irradiated human HaCaT keratinocytes. GBE exhibited scavenging activity against intracellular reactive oxygen species that were induced by either hydrogen peroxide or UVB radiation. In addition, both the superoxide anion and the hydroxyl radical were scavenged by GBE in cell-free systems. GBE absorbed light in the UVB range (280-320 nm) of the electromagnetic spectrum and lessened the extent of UVB-induced oxidative damage to cellular lipids, proteins, and DNA. Finally, GBE-treated keratinocytes showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies. These results suggest that GBE exerts cytoprotective actions against UVB-stimulated oxidative stress by scavenging ROS and absorbing UVB rays, thereby attenuating injury to cellular constituents and preventing cell death.

Fibulin-3-mediated inhibition of epithelial-to-mesenchymal transition and self-renewal of ALDH+ lung cancer stem cells through IGF1R signaling.

Fibulins (FBLNs), a family of extracellular matrix proteins, have recently been shown to act as tumor suppressors or activators in different cancers, and the underlying molecular mechanisms of their action in cancer remain unclear. We have previously shown that the expression of FBLN3 is suppressed by promoter hypermethylation and is associated with invasiveness in aggressive non-small cell lung cancer. In this study, we evaluated the roles and signaling mechanism of FBLN3 in lung cancer stem cells (CSCs). Forced expression of FBLN3 suppressed invasion and migration of lung adenocarcinoma cells and decreased the expression of epithelial-to-mesenchymal transition (EMT) activators, including N-cadherin and Snail. Stemness activities of lung adenocarcinoma cells were also suppressed by FBLN3 as indicated by a decrease in spheroid formation and the levels of stemness markers such as Sox2 and ß-catenin. These effects of FBLN3 were mediated by the glycogen synthase kinase-3ß, GSK3ß/ß-catenin pathway, and the upstream regulators of GSK3ß, including phosphoinositide 3-kinase (PI3K)/AKT and insulin-like growth factor-1 receptor (IGF1R), were inactivated by FBLN3. Moreover, IGF1R was shown to be a direct target of FBLN3, which competitively inhibited insulin-like growth factor (IGF) action. To confirm the effect of FBLN3 on lung CSCs, aldehyde dehydrogenase-positive (ALDH+) A549 lung CSCs were sorted and treated with recombinant FBLN3 protein. FBLN3 clearly suppressed EMT, stemness activity and the over-activated IGF1R/PI3K/AKT/GSK3ß pathway of the ALDH+ CSC subpopulation. In addition, injection of recombinant FBLN3 protein around subcutaneous xenografts established with ALDH+ CSCs in athymic nude mice significantly suppressed tumor growth and progression. Overall, our results show that FBLN3 suppresses both EMT and self-renewal of the lung CSCs by modulating the IGF1R/PI3K/AKT/GSK3ß pathway and that FBLN3 would be useful as an alternative CSC therapy.

Suitability of endoscopic submucosal dissection for treatment of submucosal gastric cancers.

BACKGROUND: Endoscopic submucosal dissection (ESD) is not considered appropriate for all submucosal cancers owing to the risk of lymph node metastasis and difficulty estimating the deep margin status. This study aimed to determine predictive factors for lymph node metastases in submucosal cancer and to explore in which patients ESD might be feasible. METHODS: Details of patients who had curative gastrectomy for submucosal gastric cancer at Asan Medical Centre from 2007 to 2011 were reviewed retrospectively to determine the relationship between lymph node metastasis and clinicopathological characteristics, including age, sex, tumour location, size, gross appearance, depth of invasion, histological type/differentiation, presence of lymphovascular/perineural invasion, and immunohistochemical staining results for p53, human epidermal growth factor receptor (HER) 1 and HER2. RESULTS: A total of 1773 patients were analysed. The presence of lymphovascular invasion was related most strongly to lymph node metastasis. Multivariable analysis revealed that depth of invasion, tumour size, differentiation, gross appearance and perineural invasion were also related. Metastatic lymph nodes were found in four of 105 patients who met the classical criteria for ESD; all showed a moderately differentiated histological appearance. No lymph node metastases were observed in well differentiated SM1 tumours of any size (infiltration into upper third of submucosa), or in well differentiated SM2 (infiltration into middle third of submucosa) tumours of 2 cm or less without lymphovascular invasion. CONCLUSION: Patients with well differentiated SM1 cancer of any size and those with well differentiated SM2 cancer of 2 cm or less without lymphovascular invasion may be suitable candidates for ESD.

Prognostic factors and recurrence pattern in node-negative advanced gastric cancer.

AIMS: Despite better overall survival in node-negative advanced gastric cancer (AGC), a significant proportion of patients develop recurrence and they may benefit from adjuvant therapy. The aim of this study was to evaluate the prognostic factors and recurrence pattern of node-negative AGC. METHODS: A total of 424 patients who underwent curative gastrectomy with extended lymphadenectomy for node-negative AGC between 2003 and 2005 were retrospectively reviewed. Patients with tumor involvement of adjacent organs (T4b), gastric cancer recurrence, tumor in the remnant stomach, less than 15 harvested lymph nodes, and those who received neoadjuvant chemotherapy were excluded. RESULTS: Invasion to deeper layers, undifferentiated histology, signet ring cell type compared with tubular adenocarcinoma, and tumor size larger than 6.3 cm correlated with poorer prognosis in univariate analysis. In multivariate one, however, only differentiation and depth of invasion, especially the presence of serosa involvement were significant. The 5-year survival rates of the four groups classified by differentiation and depth of invasion [T2/3 (differentiated type), T2/3 (undifferentiated type), T4a (differentiated type), and T4a (undifferentiated type)] were 98%, 92%, 80%, and 72%, respectively (P < 0.01). In terms of recurrence pattern, Lauren's type and depth of invasion were significant. Recurrence with peritoneal seeding was associated with the diffuse type and invasion into the subserosa or serosa, while hematogenous metastasis was related to the intestinal type and invasion to the proper muscle or subserosa layer. CONCLUSIONS: Differentiation and serosa involvement should be considered to stratify patients with node-negative AGC for adjuvant treatment.

In vitro activity of the EWS oncogene transcriptional activation domain.

Aberrant chromosomal fusion of the Ewings sarcoma oncogene (EWS) to several different cellular partners gives rise to the Ewing's family of oncogenic proteins [EWS fusion proteins (EFPs)] and associated tumors (EFTs). EFPs are potent transcriptional activators dependent on the N-terminal region of EWS [the EWS activation domain (EAD)], and this function is thought to be central to EFT oncogenesis and maintenance. Thus, EFPs are promising therapeutic targets, and detailed molecular studies of the EAD will be pivotal for exploring this potential. For many reasons, the molecular mechanism of EAD action is poorly understood and one major obstacle to progress is the lack of an in vitro transcription assay. Using well-characterized EAD-dependent activators and soluble nuclear extracts, we have attempted to recapitulate EAD transcriptional activity in vitro. We report that while the EAD activates transcription strongly in vitro, the effect of EAD mutations is strikingly different from that observed in vivo. Our results therefore suggest that crude soluble extracts do not support bona fide EAD activity in vitro, and we discuss our findings in relation to future assay development and potential mechanisms of EAD action.

Incomplete discoid glenoid labrum combined with a ganglion cyst of the spinoglenoid notch.

In a 41-year-old man, right-sided infraspinatus muscle weakness was associated with compression of the suprascapular nerve caused by a spinoglenoid ganglion cyst. The lesion was confirmed using electromyography and MRI. In addition, arthroscopy showed an incomplete discoid labrum. The free inner edge of the labrum was removed as in a meniscectomy of a discoid meniscus in the knee joint. Arthroscopic decompression of the cyst was performed through a juxtaglenoid capsulotomy which was left open. Neurological function recovered completely.

Salvage of the lower extremity free flap using cross-leg venous repair.

This retrospective study was designed to evaluate a salvage technique for free flaps suffering venous congestion by using a cross-leg vein repair in patients with venous-impaired lower extremities. Four free flap reconstructions were performed using the latissimus dorsi muscle to reconstruct soft tissue defects in the lower extremity. The recipient artery was confined to the ipsilateral leg and the venous anastomosis was performed with a long saphenous vein from the contralateral side. The legs were immobilised together by means of an external fixator. All patients were males with a mean age of 31 years. The mean time of pedicle division was 8.8 days (7-10). The mean size of the free flap was 186.5 cm(2). All flaps survived after pedicle division without venous congestion. There were no complications such as joint stiffness or donor site morbidity except for a linear scar. The cross-leg venous repair is a refinement of a salvage procedure for compromised free flaps used in the reconstruction of severe soft tissue defects in vascularly compromised lower extremities.

MUC1 cytoplasmic tail detection using CT33 polyclonal and CT2 monoclonal antibodies in breast and colorectal tissue.

UNLABELLED: The immunohistochemical detection (IHC) of MUC1-CT employing a polyclonal antibody (CT33) in relation to CT2 monoclonal antibody (MAb) was analyzed. Western blot (WB) was used to determine the molecular mass of CT. MATERIALS AND METHODS: We studied 163 breast and 89 colorectal cancer specimens, 10 breast and 14 colorectal benign conditions, and 12 breast and 20 colorectal normal samples. From each tumor sample, subcellular fractions were obtained and analyzed by SDS-PAGE and WB. A nonparametric statistical analysis was employed; data were standardized and a Kendall-Tau correlation was applied. RESULTS: By IHC, 146/163 (90%) and 151/163 (93%) of breast cancer were positive with CT33 and CT2, respectively; a statistically significant correlation was obtained (t=0.5199). Seven out of ten (70%) benign breast specimens were positive with CT33 while all samples stained with CT2; in normal breast sample tissues, all were positive with both Abs. In colorectal cancer samples, both antibodies stained 47/89 (53%) samples; CT2 reacted in 13/14 (93%) of benign samples while CT33 showed a positive reaction in 9/14 (64%) of benign specimens. In normal samples, CT2 showed staining in 17/20 (85%) of samples and CT33 was reactive in 12/20 (60%). By WB, in breast and colorectal cancer samples, similar results were obtained with both antibodies: a main band at about 30kDa which represents the smaller subunit. CONCLUSION: CT33 polyclonal antibody has demonstrated its efficacy to detect MUC1 in breast and colorectal cancer tissues with similar reactivity to CT2. It is worthwhile to affirm that CT33 is a good indicator of MUC1 expression.

Imatinib (ST1571) provides only limited selectivity for CML cells and treatment might be complicated by silent BCR-ABL genes.

Very promising results have been obtained in clinical trials on chronic-phase chronic myeloid leukemia (CP-CML) patients treated with imatinib mesylate (IM; Gleevecr, STI571), a BCR-ABL tyrosine kinase inhibitor. However, we found that IM caused considerable inhibition of normal hematopoietic progenitor cells upon treating control bone marrow (BM) cultures. In vitro IM treatment gave a decrease in the yield and size of colonies from BM of untreated CP-CML patients that was only two to three times that from the normal samples. Moreover, about 30% of myeloid progenitors (CFU-GM) from CML BM still formed colonies in the presence of IM, most of which had BCR-ABL RNA. About half of these treated colonies also displayed methylation of the internal ABL Pa promoter, a CML-specific epigenetic alteration, which was used in this study as a marker for BCR-ABL translocation-containing cells. However, ~5-8% of the treated or the untreated CML BM-derived colonies had no detectable BCR-ABL RNA by two or three rounds of RT-PCR despite being positive for the internal standard RNA and displaying hallmarks of CML, either t(9;22)(q34;ql 1) or ABL Pa methylation. Our results indicate that IM is only partially specific for CML progenitor cells compared to normal hematopoietic progenitor cells and suggest that some CML cells may have a silent BCR-ABL oncogene that could interfere with therapy.

Effects of polycationic peptides on mucin release from airway goblet cells: relationship between polymer size and activity.

OBJECTIVES AND DESIGN: Various sizes of poly-L-lysine (PLL) and poly-L-arginine (PLA) were tested for their possible effects on airway goblet cell mucin release using primary hamster tracheal surface epithelial (HTSE) cells in an attempt to identify the smallest size of the polycationic peptide to suppress mucin release without cytotoxicity. MATERIALS AND METHODS: HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of various sizes of the polycationic peptides. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity of the peptides was assessed by measuring the release of lactic dehydrogenase (LDH) during the treatment period. RESULTS: (1) PLL (MW 78,000) inhibited whereas PLA (MW 92,000) stimulated mucin release. However, these peptides were cytotoxic at the effective concentrations; (2) Both PLL (MW 9,600) and PLA (MW 8,900) could inhibit mucin release in a dose dependent manner without cytotoxicity; (3) Both PLL and PLA were effective in suppressing mucin release in 20-mer but not in either 10-mer or 5-mer; (4) 14-mers of both PLL and PLA also inhibited mucin release without cytotoxicity; (5) PLL and poly-D-lysine (PDL) of 14-mer were equipotent in its ability to suppress mucin release. CONCLUSION: Both PLL and PLA are cytotoxic at 'high' molecular weights, but have an ability to suppress mucin release without cytotoxicity at 'low' molecular weights. 14-mer seems to be the small, effective size, if not the smallest, for both PLL and PLA to suppress mucin release without cytotoxicity. The inhibitory effect of these polycationic peptides seems to be determined by the presence and the absolute number of positive charges and also to be independent of optical isomerism.

Amphiphysin IIb-1, a novel splicing variant of amphiphysin II, regulates p73beta function through protein-protein interactions.

p73 is a nuclear protein that is similar in structure and function to p53. Notably, the C-terminal region of p73 has a regulatory function, through interactions with a positive or negative regulator. In this study, we use the yeast two-hybrid technique to identify a novel p73beta binding protein, designated amphiphysin IIb-1. Amphiphysin IIb-1 is one of the splicing variants of amphiphysin II, and has a shorter protein product than amphiphysin IIb, which has been previously reported. We confirmed that amphiphysin IIb-1 binds full-length p73beta, both in vitro and in vivo. This association is mediated via the SH3 domain of amphiphysin IIb-1 and C-terminal amino acids 321-376 of p73beta. Double immunofluorescence patterns revealed that p73beta is relocalized to the cytoplasm in the presence of amphiphysin IIb-1. Overexpression of amphiphysin IIb-1 was found to significantly inhibit the transcriptional activity of p73beta in a dose-dependent manner. In addition, the cell death function of p73beta was inhibited by amphiphysin IIb-1. These findings offer a new insight into the regulation mechanism of p73beta, and suggest that amphiphysin IIb-1 modulates p73beta function by direct binding.

Follicular fluid enhances sperm attraction and its motility in human.

PURPOSE: Follicular fluid has a pivotal effect on motility and chemotaxis of spermatozoa for successful fertilization. The effect of human follicular fluid (hFF) and progesterone on attraction and motility of spermatozoa were investigated using simplified capillary assays. METHODS: Capillary tubes loaded with hFF, modified human tubal fluid (m-hTF), or m-hTF supplemented with progesterone, respectively, were used for assessments of attraction and motility of spermatozoa following culture at various time intervals. RESULTS: Number and motile ratio of spermatozoa in the tubes loaded with hFF were significantly (P < .05) higher than those with m-hTF. In the tubes loaded with m-hTF, m-hTF supplemented with progesterone, and hFF, the attracted number of spermatozoa were 34 x 10(5), 131 x 10(5), and 108 x 10(5), and motile ratio of spermatozoa was 37, 48, and 82%, respectively. CONCLUSIONS: We conclude that hFF clearly plays a crucial role in enhancing attraction and motility of spermatozoa, and progesterone has strong effect on attraction of spermatozoa.

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant.

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.