Yoshitaka Hishikawa

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation.

Expression of Fas and Fas Ligand in Normal and Ischemia-Reperfusion Testes: Involvement of the Fas System in the Induction of Germ Cell Apoptosis in the Damaged Mouse Testis

Abstract Apoptosis of germ cells is very common in normal and injured mammalian testes. The aim of this study was to examine the possible involvement of the Fas and Fas ligand (FasL) system in the induction of germ cell apoptosis in normal and ischemia-reperfusion testes of adult mice. Apoptosis was assessed by the TUNEL method and by DNA gel electrophoresis. Fas and FasL mRNAs were detected by Northern blotting and reverse transcription polymerase chain reaction techniques, and proteins were analyzed by Western blotting and immunohistochemistry. Apoptosis of germ cells was identified in the normal testis especially around stages XI and XII, whereas the expression of Fas and FasL was largely confined to Leydig cells and Sertoli cells, respectively. However, in the testes reperfused after 1 h of ischemia, a high number of TUNEL-positive cells were identified in parallel with increased Fas-positive germ cells, whereas FasL expression in Sertoli cells was almost constant irrespective of the duration of reperfusion. Moreover, i.p. injection of anti-Fas antibody, which blocks the interaction between Fas and FasL, inhibited apoptosis, as indicated by the reduced number of TUNEL-positive cells, except for apoptosis at stages XI and XII. Our results indicate that the Fas/FasL system mediates apoptosis of spermatogenic cells in the injured testis but not spontaneous apoptosis in the normal testis.

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis.

Metallothionein expression correlates with metastatic and proliferative potential in squamous cell carcinoma of the oesophagus

The goal of this study is to clarify whether the expression of metallothionein (MT) could affect the prognosis and the metastatic potential of squamous cell carcinoma (SCC) of the oesophagus. In paraffin-embedded specimens resected from 57 patients, MT mRNA and protein expressions were detected by in situ hybridization and immunohistochemistry respectively. The expression of MT was evaluated in respect of clinicopathologic variables and patients’ survival. MT mRNA expression was significantly associated with the proportion of lymph node metastasis (71% in MT mRNA-positive tumours vs 42% in MT mRNA-negative tumours; P = 0.0343) and that of distant metastasis (29% in MT mRNA-positive tumours vs 5% in MT mRNA-negative tumours; P = 0.0452). In respect of MT protein expression, the frequency of distant metastasis was more common in MT-positive tumours than in MT-negative tumours (30% in MT-positive tumours vs 8% in MT-negative tumours; P = 0.0446). The survival rate of the patients with MT protein-negative tumours was significantly better than that of the patients with MT protein-positive tumours (P = 0.0340). There was a positive correlation between the expression of MT protein and that of proliferating cell nuclear antigen (P = 0.0018). Therefore, we conclude that MT expression, both at the mRNA and protein levels, may be a potential marker predicting metastatic and proliferative activities of oesophageal SCC.

Prognostic significance of perioperative blood transfusions in resectable thoracic esophageal cancer

OBJECTIVE:The perioperative blood transfusions have been associated with tumor recurrence and decreased survival in various types of alimentary tract cancer. There exist, however, contradictory studies showing no relationship between blood transfusions and survival. For patients with esophageal cancer, only one report suggested that blood transfusions did not by itself decrease the chance of cure after esophagectomy.METHODS:Among 235 patients with primary squamous cell carcinoma of the thoracic esophagus between December 1979 and March 1998, 143 patients (60.9%) underwent esophagectomy with curative intent (RO). To exclude the effects of surgery-related postoperative complications, 14 patients who died within 90 days during the hospital stay were excluded. Thus, clinicopathological characteristics and prognostic factors were retrospectively investigated between patients with no or few transfusions (≤2 units) (n = 58), and much transfused patients (≥3 units) (n = 71).RESULTS:Sixty-three patients are alive and free of cancer, and 66 patients are dead. A total of 98 patients (76%) received blood transfusions, whereas 31 patients (24%) had no transfusion. The amount of blood transfused was 1 or 2 units in 27 patients (27.6%), 3 or 4 units in 33 (33.7%), 5 or 6 units in 20 (20.4%), and ≥7 units in 18 (18.4%). The 5-yr survival rate for patients with no or few transfusions was 69%, whereas that for much transfused patients was 31.7% (p < 0.0001). The much transfused patients had more prominent ulcerative tumor, longer time of operation, more estimated blood loss, and more marked blood vessel invasion than the group with no or few transfusions. The factors influencing survival rate were tumor location, Borrmann classification, size of tumor, depth of invasion, number of lymph node metastases, time of operation, amount of blood transfusions, lymph vessel invasion, and blood vessel invasion. Among those nine significant variables verified by univariate analysis, independent prognostic factors for survival determined by multivariate analysis were number of lymph node metastasis (0 or 1 vs≥2, p < 0.0001), amount of blood transfusions (≤2 units vs≥3 units, p < 0.0001), and blood vessel invasion (marked vs non-marked, p= 0.0207).CONCLUSIONS:There is an association between high amount of blood transfusions and decreased survival for patients with resectable esophageal cancer. To improve the prognosis, surgeons must be careful to reduce blood loss during esophagectomy with extensive lymph node dissection and subsequently must minimize blood transfusions.

Prognostic Significance of the Expressions of Metallothionein, Glutathione-S-Transferase-π, and P-Glycoprotein in Curatively Resected Gastric Cancer

Although experimental studies indicate that overexpression of metallothionein (MT), glutathione-S-transferase-pi (GST-pi), or P-glycoprotein (P-GP) is related to the drug resistance of cancer cells, the clinical significance of the overexpression remains to be elucidated. The expressions of MT, GST-pi, and P-GP wre evaluated immunohistochemically in 74 specimens of gastric adenocarcinoma in T1-3N1-2 stages which were resected with curative intent. Fluorinated pyrimidines, mitomycin C, and Adriamycin were prescribed in 73, 54, and 2 patients, respectively. The staining characteristics were investigated in relation to the clinical results. The cell-proliferative activity was studied with anti-proliferating cell nuclear antigen antibody. Expressions of GST-pi and P-GP correlated with the staining intensity of normal mucosa. Five-year disease-free survival rates (DFSRs) of GST-pi-negative and GST-pi-positive groups were 75.0 and 49.0%. The 5-year DFSRs of P-GP-negative and P-GP-positive groups were 68.2 and 38.6%. Concurrent expression among the three proteins was associated with the survival: 5-year DFSR of no- or one-protein-positive group was 75.0%, while those of 2- and 3-protein-positive groups were 56.0 and 38.9%, respectively. Tumors concurrently expressing 2 or 3 proteins have a high proliferative activity. Expressions of MT, GST-pi, and P-GP by the tumor are associated with a poorer prognosis of the patients.

Identification and localization of estrogen receptor α- and β-positive cells in adult male and female mouse intestine at various estrogen levels

Although estrogen is implicated in the regulation of mammalian intestinal function, the presence and the distribution of estrogen receptor (ER)-positive cells in the intestine are still controversial. The present study was designed to localize ERα- and ERβ-expressing cells in female and male mouse intestines immunohistochemically under various estrogen conditions, especially in female mice, ovariectomized as well at various phases of the estrous cycle. Western blot analysis detected both ERα (66-kDa band) and ERβ (56-kDa band). Immunohistochemical staining of paraffin-embedded sections after antigen-retrieval treatment with autoclaving revealed staining for ERα in submucosal interstitial cells, and double staining identified these cells as a subtype of intestinal macrophages. The number of these cells varied according to the estrous cycle phase. Administration of 17β-estradiol to ovariectomized mice resulted in a significant increase in the number of ERα-positive macrophages. On the other hand, the nuclei of nerve cells in Auerbach and Meissner plexuses were positive for both ERα and ERβ, but the number of positive nerve cells was not affected by estrogen. Our results indicate that estrogen and estrogenic compounds may exert their actions on the intestine in two ways; one is through interstitial macrophages and the other is through intestinal neurons.

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T–T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) α and β, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER α, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER α-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER α may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.

Transforming Growth Factor-β1 Released from the Spleen Exerts a Growth Inhibitory Effect on Liver Regeneration in Rats

Several previous reports indicated that partial hepatectomy (PH) when combined with splenectomy enhances the regenerative capacity of the liver, most probably due to the removal of unknown inhibitory factors released from the spleen. Transforming growth factor (TGF)-β1 is a major antiproliferative factor for the hepatocytes, and the role of splenic TGF-β1 in liver regeneration is yet to be clarified. The splenic expression of TGF-β1 and its secretion into the portal circulation from the spleen were evaluated in a standardized two-thirds hepatectomy model in rats. Rats in the control group underwent only the hepatectomy, while splenectomy was added in the splenectomy group. The hepatocyte proliferation rate was assessed by proliferating cell nuclear antigen (PCNA) immunostaining, and the results were compared with the TGF-β1 kinetics in the portal blood. Significant increase in PCNA index and decrease in portal TGF-β1 level were noticed in the splenectomy group at 48 hours after PH compared with the control group. Both TGF-β1 protein and mRNA expression level in the spleen were strongest at 48 hours after PH and coincided with the peak of the plasma TGF-β1 level. TGF-β type II receptor (TβR-II) expression in the liver after PH was assessed immunohistochemically. The expression of TβR-II decreased at 12 hours and returned to preoperative level at 24 hours after PH in both groups. The changes of TβR-II expression were similar in both groups, and the significant difference was not observed at 48 hours after PH. Namely, splenectomy did not alter the expression of TβR-II in remnant liver at the peak of hepatocytes proliferation. In conclusion we found that TGF-β1 was produced and secreted by the spleen during the early phase of liver regeneration and removal of the spleen enhanced proliferation of hepatocytes. Splenectomy thus may exert a salutary effect in selected patients with jeopardized regenerative capacity of the liver.

Possible Involvement of Keratinocyte Growth Factor and Its Receptor in Enhanced Epithelial-Cell Proliferation and Acquired Recurrence of Middle-Ear Cholesteatoma

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.