Effects of supplemental oxygen on urinary 8-hydroxy-2'-deoxyguanosine levels in extremely low birth weight infants.

As the effects of supplementary oxygen on urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) are poorly understood, urinary 8-OHdG levels (ng/mg creatinine) were determined longitudinally on the postnatal day (PND) 1, 3, and 30 in 16 neonates with birth weight < 1000 g. No supplementary oxygen was required in 9 neonates during the first 24 h of life. Urinary 8-OHdG level on PND 1 was inversely correlated with birth weight in these 9 neonates (P = 0.0323) and was higher in four with birth weight < 750 g than five with birth weight > 750 g (41.0 ± 6.9 vs. 5.6 ± 2.7, respectively, P = 0.0200). Median urinary 8-OHdG on PND 1 of these 9 neonates was significantly lower than that of 7 neonates with oxygen (9.3 vs. 60.2, respectively), although there were no significant differences in clinical background, such as birth weight, between the two groups. Five of the 9 did not require supplemental oxygen at all during the first 30 days of life. Median urinary 8-OHdG levels were consistently significantly lower in the 5 neonates than in 11 neonates with oxygen transiently or persistently (9.3 vs. 54.6, 19.1 vs. 61.4, and 28.3 vs. 145 on PND 1, 3, and 30, respectively), although there were no differences in clinical background, such as birth weight, between the two groups. Urinary 8-OHdG on PND 30 was significantly positively correlated with supplemental oxygen dose on PND 30 (P < 0.0001), but not with birth weight in the 16 neonates. These results suggest that higher supplemental oxygen tension caused higher urinary 8-OHdG in this population.

Plant homeodomain finger protein 11 promotes class switch recombination to IgE in murine activated B cells.

BACKGROUND: Polymorphisms of the Plant homeodomain finger protein 11 (PHF11) are strongly associated with high serum IgE levels and clinical severity of atopic patients. However, the precise mechanism has not been fully elucidated. We investigated the role of Phf11 in class switch recombination (CSR) to IgE by activated B cells. METHODS: We generated Phf11 transgenic (Lckd-Phf11-Tg) mice that express the exogenous murine Phf11 in lymphocytes under the control of distal Lck promoter. We examined IL-4-induced CSR to IgE in activated Lckd-Phf11-Tg B cells in vitro. We analyzed production of ovalbumin (OVA)-specific IgE and nose-scratching symptoms in Lckd-Phf11-Tg mice using an OVA-induced allergic rhinitis model. RESULTS: The exogenous Phf11 promoted CSR to IgG1 and IgE in activated B cells with an increase in germ line transcript (GLT) γ1 and GLT ε expression. The exogenous Phf11 augmented transcriptional activity of the GLT γ1 and GLT ε promoters through permissive histone modifications and binding of NF-κB and STAT6. Furthermore, the exogenous Phf11 bound to the GLT ε promoter with increased binding of NF-κB. Silencing of the endogenous Phf11 reduced the frequency of CSR to IgE and GLT ε expression, but not to IgG1 or GLT γ1 expression, in activated B cells. In an allergic rhinitis model, Lckd-Phf11-Tg mice showed a significant increase in the production of OVA-specific IgE and the frequency of nose scratching. CONCLUSION: Phf11 accelerates CSR to IgE in activated B cells by increasing the transcriptional activity of GLT ε promoter and contributes to the exacerbation of allergic responses. These findings provide a novel therapeutic target for allergic diseases.

Bcl6 in pulmonary epithelium coordinately controls the expression of the CC-type chemokine genes and attenuates allergic airway inflammation.

BACKGROUND: There is synteny in the CC-type chemokine gene clusters between humans (CCL2/MCP-1, CCL7MCP-3, CCL11/eotaxin, CCL8/MCP-2, CCL13/MCP-4, and CCL1/I-309) and mice (CCL2, CCL7, CCL11, CCL12/MCP-5, CCL8, and CCL1). OBJECTIVE: As many putative Bcl6/STAT-binding sequences are observed in the clusters, we examined the roles of a transcriptional repressor Bcl6 and the regional histone modification in the expression of these chemokine genes in pulmonary epithelium. METHODS: We generated transgenic (Tg) mice carrying the Bcl6 or the dominant-negative (DN)-Bcl6 gene under the control of the surfactant protein C (SPC) promoter that induces the exogenous gene expression in the distal lung epithelium. For in vitro studies, A549, alveolar type II-like epithelial cell line transfected with the SPC-DN-Bcl6 gene were stimulated with IL-4+TNF-α, and Bcl6 or STAT6 binding to and histone modification of the cluster in the transfectants were analysed by chromatin immunoprecipitation assays. Tg mice sensitized with ovalbumin (OVA) were challenged with OVA inhalation. The amounts of mRNAs in each sample were analysed by quantitative RT-PCR. RESULTS: The amount of Bcl6 bound to the cluster decreased in A549 cells stimulated with IL-4 and TNF-α, whereas STAT6 binding increased in association with regional histone H3-K9/14 acetylation and H3-K4 methylation. The expression of all chemokine genes in the gene cluster was augmented in activated A549 cells transfected with the DN-Bcl6 gene. We also induced allergic airway inflammation in Tg mice. Expression of the chemokine genes and infiltrated cell numbers in the lungs of these Tg mice with allergic airway inflammation were inversely correlated with the amount of Bcl6 in the lungs. CONCLUSION AND CLINICAL RELEVANCE: Expression of the pulmonary epithelium-derived CC-type chemokine genes in the cluster is orchestrated by the conserved machinery related to Bcl6. Thus, Bcl6 in pulmonary epithelium may be a critical regulator for pathogenesis of various pulmonary inflammatory diseases.

Over-expression of the LTC4 synthase gene in mice reproduces human aspirin-induced asthma.

BACKGROUND: The pathogenesis of aspirin-induced asthma (AIA) is presumed to involve the aspirin/non-steroidal anti-inflammatory drug (NSAID)-induced abnormal metabolism of arachidonic acid, resulting in an increase in 5-lipoxygenase (5-LO) metabolites, particularly leukotriene C(4) (LTC(4) ). However, the role of LTC(4) in the development of AIA has yet to be conclusively demonstrated. OBJECTIVE: The aim of this study was to evaluate the contribution of the lipid product LTC(4) secreted by the 5-LO pathway to the pathogenesis of AIA. METHODS: To evaluate antigen-induced airway inflammation, the concentrations of T-helper type 2 cytokine in bronchoalveolar lavage fluid (BALF) obtained from LTC(4) synthase-transgenic (Tg) and wild-type (WT) mice after challenge with ovalbumin were measured. Subsequently, the ex vivo and in vivo effects of the NSAID sulpyrine were investigated in these Tg and WT mice by measuring the secretion of LTC(4) from sulpyrine-treated BAL cells and the levels of LTC(4) in BALF following challenge with sulpyrine. Finally, the sulpyrine-induced airway response by the administration of pranlukast, an antagonist of the cysteinyl (cs)-LT1 receptor, was analysed. RESULTS: The concentrations of IL-4, -5, and -13 in BALF from Tg mice were significantly higher than those in WT mice. In addition, sulpyrine augmented the secretion of LTC(4) in BALF and by BAL cells in Tg mice, but not in WT mice. Additionally, the increased airway resistance induced by sulpyrine could be reduced by treatment with pranlukast. Furthermore, the secretion of LTC(4) from mast cells, eosinophils, and macrophages was increased in the allergen-stimulated LTC(4) synthase gene Tg mice, even in the absence of sulpyrine, as well as in BAL cells after sulpyrine. CONCLUSION AND CLINICAL RELEVANCE: The over-expression of the LTC(4) synthase in a mouse asthma model also replicates the key features of AIA. And our study supports that cys-LTs play a major role in the pathogenesis of AIA in patients with chronic asthma.

Radiographic findings of diaphragmatic hernia and hypoplastic lung.

OBJECTIVE: Congenital diaphragmatic hernia (CDH) has a poor prognosis, despite intensive management. The prognosis of CDH is correlated with hypoplastic lung, but it is difficult to measure the degree of hypoplasia. The aims of this study were, therefore, to examine the relationship between chest X-ray and prognosis, and to assess whether the radiographic findings were a good indicator of hypoplastic lungs in patients with CDH. STUDY DESIGN: Fifty neonates with CDH were classified radiographically into apex and hilar types. To assess the differences in clinical course between these two groups, gestational age, birth weight, prenatal diagnosis, survival rate, requirement of extracorporeal membrane oxygenation (ECMO) therapy and lung area on X-rays were analyzed. RESULTS: In all, 32 cases were of the apex type and 18 were hilar. The survival rate of the hilar group (33%) was significantly worse than that of the apex group (81%) (P<0.001). The hilar group required ECMO therapy more frequently than did the apex group. CONCLUSIONS: The present results show a significant correlation between survival rate and the findings of chest X-rays in CDH. Radiographic findings are thus a good clinical indicator of the prognosis of CDH in neonates.

Isoform-dependent interaction of BRDG1 with Tec kinase.

Tec is the prototype of an emerging family of protein-tyrosine kinases. Tec and Btk, another member of this family, together participate in the development of B-cell immune system. We previously identified one of the downstream messengers for human Tec kinase, BRDG1. BRDG1 is associated with Tec and becomes tyrosine-phosphorylated in B-cells by the engagement of B-cell antigen receptor (BCR). Here we show that overexpression of BRDG1 strongly augments BCR-mediated activation of cAMP-response element binding protein (CREB) but not that of c-Jun and the promoters of c-MYC and BCL-xL genes. Furthermore, we isolated the murine orthologue of BRDG1. Three isoforms of BRDG1 are generated by alternative splicing of the message. Two of them have a deletion of 33 amino acids in a Pleckstrin homology (PH) domain of BRDG1. Both the tyrosine-phosphorylation and CREB-activating ability of BRDG1 were isoform-dependent, suggesting a role of the PH domain of BRDG1. These data have identified a novel regulatory mechanism of CREB family of transcriptional factors.

A new functional domain of Bcl6 family that recruits histone deacetylases.

The proto-oncogene Bcl6 and its family gene, BAZF, encode a sequence-specific transcriptional repressor which contains the BTB/POZ domain in NH(2)-terminal region and zinc finger motifs in COOH-terminal region. The BTB/POZ domain and the middle portion of Bcl6 and BAZF are known to display transrepressor activity. Since we have identified the identical 17-amino acid (aa) sequence in the middle portion of Bcl6 and BAZF, the 17aa region may be another repressive domain of the middle portion. The reporter gene assay indicates that the 27aa sequence including the 17aa region recruits histone deacetylases to express transrepressor activity. Furthermore, overexpression of Bcl6 or Bcl6(POZ-) (Bcl6 deleted with the BTB/POZ domain) induced apoptosis in NIH3T3 cells, and the apoptosis was inhibited by the addition of histone deacetylase inhibitor in the culture. However, apoptosis was not induced in NIH3T3 cells by overexpression of Bcl6(POZ-) deleted with the 17aa region. These results indicate that the 17aa region in the middle portion of Bcl6 is a functional domain of transrepressor activity and is responsible for inducibility of apoptosis in NIH3T3 cells.

Nd1, a novel murine Kelch family protein, may play the role of a housekeeping gene.

The murine Nd1 gene encodes a novel Kelch family protein and expresses two forms of mRNA, long (Nd1-L) and short (Nd1-S), in various tissues. We characterized the genomic organization of the Nd1 gene, and found that Nd1-L and Nd1-S consist of 16 and nine exons respectively, and that exons I-VIII are shared between them. Three transcription initiation sites were identified in the 5'-flanking region and the most 3' side (+1) is likely to be a major one. Promoter analysis revealed that the region between positions -247 and -86 was sufficient for expression, and that two Sp1-binding sites and one NF-kappaB-binding site in the region were critical for promoter activity. Furthermore, the promoter region lacks a TATA and a CAAT box and has a highly GC-rich region with two important Sp1-binding sites. These characteristics of the Nd1 gene promoter are similar to the properties of housekeeping genes.

Binding of BAZF and Bc16 to STAT6-binding DNA sequences.

BAZF, a family member of Bcl6, can function as a sequence-specific transcriptional repressor. We determined BAZF-binding DNA sequence. The consensus binding sequence (CBS) of BAZF is almost the same as those of Bcl6 previously described. Three nucleotides of T, G and A at position 6, 8, and 9 in the CBS (5'-ATTCCTAGAAAG-3') are important nucleotides for binding of both BAZF and Bcl6. Since a part (5'-TTC-CTA-GAA-3') of the CBS resembled the sequence motif (5'-TTC-(N3-4)-GAA-3') bound by STAT factors, BAZF and Bcl6 can bind to the CD23b-STAT6-binding sequence (5'-TTTC-TTA-GAAAT-3'), the immunoglobulin germline epsilon-STAT6-binding sequence (5'-CTTC-CCAA-GAAC-3'), and the IL4-STAT6-binding sequence (5'-TTTC-CCA-GAAAA-3') with weak affinity. However, a mutation of C nucleotide to T nucleotide in the IL4-STAT6-binding sequence (5'-TTTC-CTA-GAAAA-3') strongly increased the binding activity of BAZF and Bcl6. These results suggest that BAZF and Bcl6 can repress some of STAT-induced transcription by binding to DNA sequences recognized by STAT factors.

A novel homologue of the TIAP/m-survivin gene.

The inhibitor of apoptosis (IAP) proteins comprise a highly conserved gene family that prevents cell death in response to a variety of stimuli. TIAP/m-survivin, a murine homologue of human Survivin, is a member of the IAP family. TIAP/m-survivin has one baculovirus IAP repeat (BIR) and lacks a C-terminal RING finger motif. Here we identified the genomic DNA region (TIAP-2) that is homologous to the TIAP/m-survivin gene by a low stringency genomic DNA hybridization. The region is on the chromomsome 9 which is distinct from that (chromosome 11) of the TIAP/m-survivin gene, and contains DNA sequence similar to a part of the BIR and the 3' side of the TIAP/m-survivin gene and the sequence homology between them is 92%. Expression of TIAP-2 mRNA was detected in various murine tissues by RT-PCR. Although expression of TIAP/m-survivin mRNA is upregulated in synchronized cells at S to G2/M phase of the cell cycle, expression of TIAP-2 mRNA was constant in the cell cycle, suggesting the different role of TIAP-2 from that of TIAP/m-survivin.

Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

Vesicourethral sphincter dysfunction in ncx deficient mice with an increased neuronal cell number in vesical ganglia.

PURPOSE: Ncx/Hox11L.1 knockout mice have a megacolon with an increased number of neuronal cells in the enteric ganglia. Since Ncx/Hox11L.1 is expressed in neuronal cells in the vesical ganglia, we examined lower urinary tract function and the number of neuronal cells in the vesical ganglia in Ncx/Hox11L.1 knockout mice. METHODS: Female knockout and control mice were investigated in regard to voiding frequency, and cystometry and histological studies were done. The number of neuronal cells in the vesical ganglia was observed by staining with nicotinamide adenine dinucleotide phosphate diaphorase and cuprolinic blue. RESULTS: In knockout mice voiding frequency was 2-fold and bladder capacity was less than in controls. Although bladder structure was histologically similar in knockout mice and controls, cystometry showed that threshold and remaining pressure was less in knockout mice. Neuronal cells positive for nicotinamide adenine dinucleotide phosphate diaphorase or cuprolinic blue were more numerous in the vesical ganglia of knockout mice than controls. The intraperitoneal injection of a nitric oxide synthase inhibitor increased threshold and remaining pressure on cystometry in knockout mice to the control level. CONCLUSIONS: The increased number of neuronal cells in the vesical ganglia induces vesicourethral sphincter muscle dysfunction in knockout mice. Since administering a nitric oxide synthase inhibitor somewhat overcomes the dysfunction, the amount of nitric oxide in vesical nerve cells is important for controlling vesicourethral sphincter muscle function.

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity.

The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes, and the majority of CD4(-)CD8(+) and CD4(-)CD8(-) [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP(+) cells was detected in non-T lineage subsets, including mature and immature B cells, CD5(+) B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP(+) cells detected were found in the CD44(+)CD25(-) DN thymocyte subpopulation. The developmental potential of GFP(-) and GFP(+) cells in the CD44(+)CD25(-) DN fraction was examined using in vitro culture systems. The generation of substantial numbers of alphabeta and gammadelta T cells as well as NK cells was demonstrated from both GFP(-) and GFP(+) cells. However, no development of B cells or dendritic cells was detected from GFP(+) CD44(+)CD25(-) DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44(+)CD25(-) DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.

Testicular germ cell apoptosis in Bcl6-deficient mice.

Bcl6 protein has been detected in testicular germ cells, mainly spermatocytes, of normal mice, but its physiological role is largely unknown. The number of spermatozoa in the cauda epididymis of adult Bcl6-deficient (Bcl6-/-) mice is lower than that of Bcl6+/+ mice. We have found numerous apoptotic spermatocytes at the metaphase I stage with induction of Bax protein in adult Bcl6-/- testes. Developmentally, the incidence of germ cell apoptosis of Bcl6-/- mice was similar to that of Bcl6+/+ mice until six weeks of age and increased after eight weeks of age. The incidence of apoptosis in heterozygous Bcl6+/- mice was also higher than that of Bcl6+/+ mice. Since the activated form of p38 MAP kinase was detected in spermatocytes of adult Bcl6-/- mice, the germ cell apoptosis may be induced by stressors. Treatment of testes of adult Bcl6+/+ mice with a mild hyperthermia resulted in germ cell apoptosis predominantly in metaphase I spermatocytes with induction of Bax protein and activation of p38 MAP kinase and this apoptosis mimics that in adult Bcl6-/- mice. Thus, Bcl6 may play a role as a stabilizer in protecting spermatocytes from apoptosis induced by stressors.

Tissue-specific expression of a suicide gene for selective killing of neuroblastoma cells using a promoter region of the NCX gene.

The human NCX gene, a homologue of the murine neural crest homeobox (Ncx/Hox11L.1) gene whose expression is restricted to a subset of neural crest-derived tissues, was expressed in human neuroblastoma cells but not in other tumors or fibroblasts. A 4.5-kb genomic fragment in the 5'-flanking region of the NCX gene efficiently transcribed the fused luciferase reporter gene in human neuroblastoma cells but not in non-neuroblastoma cells. Sequential deletion of this regulatory region from the 5' side demonstrated that a 1.7-kb fragment upstream from the start codon retained the preferential promoter activity in neuroblastoma cells. The transcriptional activation by the NCX promoter was stronger than that by the SV40 T antigen promoter in human neuroblastoma cells. Transfection of neuroblastoma cells with the NCX promoter-linked herpes simplex virus-thymidine kinase (HSV-TK) gene increased their sensitivity to ganciclovir. The regulatory region of the NCX gene is thus useful for neuroblastoma-specific suicide gene therapy.

Transcriptional regulation of memory B cell development.

Abstract Antigen-reactive B cells in the spleen of mice immunized with T cell-dependent antigens generate antibody-producing foci in periarteriolar lymphoid sheaths (PALS) or migrate into follicles to form germinal centers. Germinal center B cells clonally expand, have somatic hypermutation in IgV-region genes, are selected by apoptosis on the basis of antigen-specific signals, and differentiate to memory B cells. Two transcription factors (Bcl6 and c-Fos) in B cells play a critical role in the development of germinal centers. (1) Bcl6 is highly expressed in germinal center B cells, and defects in B cells perturb the formation of germinal centers but not that of PALS-associated foci, indicating the essential role of Bcl6 in the differentiation. (2) Overexpression of c-Fos in germinal center B cells induces apoptosis and perturbs the formation of memory B cells. Overexpression of Bcl-2 cannot rescue c-Fos-induced apoptosis in germinal center B cells. Since c-Fos is induced in mature B cells which have reacted with antigens, and clonal deletion of self-reactive B cells is insensitive to overexpression of Bcl-2, c-Fos may play a causal role in the clonal deletion of germinal center B cells. Thus, these factors provide a unique opportunity to investigate the molecular mechanisms of memory B cell development.

Relation of estrogen receptor expression in endometrial cancer specimens to bone mineral density.

BACKGROUND: To investigate the relation of estrogen and progesterone receptor (ER and PR) expression in endometrial cancer specimens to bone mineral density (BMD). MATERIALS AND METHODS: Subjects were 48 postmenopausal women with endometrial cancer treated with hysterectomy. Baseline characteristics included age, years since menopause (YSM), height, weight, and body mass index (BMI). Lumbar spine BMD (L2-4), the ratio of trunk fat to leg fat mass (trunk-leg fat ratio), body fat mass, and the percentage of body fat were measured by dual-energy x-ray absorptiometry (DEXA). ER and PR expression in endometrial cancer specimens were determined immunohistochemically. RESULTS: Of 48 women, 32 (66.7%) were ER/PR-positive, nine (18.8%) were ER/PR-negative, three (6.3%) were ER-positive/PR-negative, and four (8.3%) were ER-negative/PR-positive. Lumbar spine BMD and trunk-leg fat ratio were significantly higher in women with ER-positive than in those with ER-negative (p<0.05), but other variables did not differ between the two groups. BMD and baseline- and anthropometric characteristics did not differ between the two groups divided by the presence or absence of PR expression. CONCLUSIONS: ER expression in endometrial cancer specimens is associated with higher lumbar spine BMD.

HCV-core protein accelerates recovery from the insensitivity of liver cells to Fas-mediated apoptosis induced by an injection of anti-Fas antibody in mice.

BACKGROUND/AIMS: Hepatitis C virus (HCV) is a major etiologic agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The aim of this study was to elucidate pathological effects of HCV-core protein on liver cells. METHODS: We have generated transgenic mice carrying HCV-core cDNA (Px-core) and pathologically examined livers of Px-core mice. RESULTS: HCV-core protein was detectable in livers from lines 5 (C5) and 8 (C8) of Px-core transgenic mice. Since chronic hepatitis and cirrhosis precede hepatocellular carcinoma in patients with HCV infection, we tried to examine the effect of repetitive injection of a small dose of anti-Fas antibody in the transgenic mice. Surprisingly, an initial injection of anti-Fas antibody induced resistance of liver cells to the second injection of anti-Fas antibody in both Px-core and littermate control mice. The insensitivity of liver cells induced in the control mice continued for more than 24 weeks after the first injection but was broken within 1 week after partial hepatectomy. However, the sensitivity was restored in the Px-core mice within 12 weeks after the injection. CONCLUSION: HCV-core protein in liver cells may affect persistence of Fas-mediated liver cell injury.

Cell cycle-dependent regulation of TIAP/m-survivin expression.

TIAP, a murine homologue of human survivin, is a member of the inhibitor of apoptosis (IAP) family and is specifically expressed at G2/M phase of the cell cycle. To elucidate regulatory mechanisms of the cycle-dependent expression, we have analyzed the promoter region of TIAP/mouse survivin (m-survivin). The 5'-flanking region of the TIAP/m-survivin gene contained a TATA-less promoter, two AP2 sites, three NF-kB sites, one Sp1 site, many cell cycle-dependent elements (CDEs) and one cell cycle gene homology region (CHR). Primer extension and 5'-rapid amplification of cDNA ends identified one transcription start site at position -100 upstream of the ATG start site (+1). TIAP/m-survivin promoter-luciferase analysis identified a minimal promoter region within the most proximal -271 bp upstream of the ATG start site, and the region between -410 and -272 was critical for the enhancer activity. The combination between the CHR at -51 and the CDE at -57 is also essential for the cell cycle-dependent expression. Mutation of the CDE/CHR element and the enhancer elements may cause disordered expression of TIAP/m-survivin to affect cell survival and oncogenesis.