A combinatorial code for gene expression generated by transcription factor Bach2 and MAZR (MAZ-related factor) through the BTB/POZ domain.

Bach2 is a B-cell- and neuron-specific transcription repressor that forms heterodimers with the Maf-related oncoproteins. We show here that Bach2 activates transcription by interacting with its novel partner MAZR. MAZR was isolated by the yeast two-hybrid screen using the BTB/POZ domain of Bach2 as bait. Besides the BTB/POZ domain, MAZR possesses Zn finger motifs that are closely related to those of the Myc-associated Zn finger (MAZ) protein. MAZR mRNA was coexpressed with Bach2 in B cells among hematopoietic cells and in developing mouse limb buds, suggesting a cooperative role for MAZR and Bach2 in these cells. MAZR forms homo- and hetero-oligomers with Bach2 through the BTB domain, which oligomers bind to guanine-rich sequences. Unlike MAZ, MAZR functioned as a strong activator of the c-myc promoter in transfection assays with B cells. However, it does not possess a typical activation domain, suggesting a role for it as an unusual type of transactivator. The fgf4 gene, which regulates morphogenesis of limb buds, contains both guanine-rich sequences and a Bach2 binding site in its regulatory region. In transfection assays using fibroblast cells, the fgf4 gene was upregulated in the presence of both MAZR and Bach2 in a BTB/POZ domain-dependent manner. The results provide a new perspective on the function of BTB/POZ domain factors and indicate that BTB/POZ domain-mediated oligomers of transcription factors may serve as combinatorial codes for gene expression.

Expression of metalloproteinase-13 (Collagenase-3) is induced during fracture healing in mice.

In fracture healing, a large amount of cartilage is formed, then rapidly replaced by osseous tissue. This process requires the transition of extracellular matrix component from type II to type I collagen. We investigated the expression of matrix metalloproteinase-13 (MMP-13), which has a high potential to cleave type II as well as type I collagen, during fracture repair in mouse ribs. In situ hybridization demonstrated that MMP-13 mRNA was present throughout the healing process. It was detected in the cells of the periosteum at day 1. As fracture callus grew, strong MMP-13 mRNA signals were detected in cells of the cartilaginous callus. In the reparative and remodeling phases, both hypertrophic chondrocytes and immature osteoblastic cells in the fracture callus expressed MMP-13 mRNA strongly. These cells were located adjacent to tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts at the sites of cartilage/bone transition. In osteoclasts, MMP-13 expression was not detected. The level of MMP-13 mRNA peaked at day 14 postfracture by northern blotting. Immunohistochemical staining showed that MMP-13 was detected primarily in hypertrophic chondrocytes. These results indicate that MMP-13 is induced during fracture healing. The site- and cell-specific expression of MMP-13 and its enzymatic property suggest that MMP-13 initiates the degradation of cartilage matrix, resulting in resorption and remodeling of the callus. In conclusion, MMP-13 plays an important role in the healing process of fractured bone in mice.

Effects of aging on hydroxyproline in human heart muscle.

Hydroxyproline concentration in the NaCl-soluble, TCA-soluble, and residual fractions of heart muscle was chemically determined in 40 autopsy subjects (25 men and 15 women), to study the effects of aging. In the left ventricle, the total hydroxyproline content of the endocardium and papillary muscle increased significantly in relation to age; the increase was greatest in the TCA-soluble fraction, followed by the residual fraction. Statistical analysis revealed that hydroxyproline in the residual fraction increased from the epicardium to the endocardium and then to the papillary muscle. Significant negative correlations were found between heart weight and total hydroxyproline in the epicardium and endocardium. In the right ventricle, a significant negative correlation was noted between heart weight and total hydroxyproline content. Though no correlations were evident between age and hydroxyproline content in females, significant correlations were observed between age and the total hydroxyproline content of the endocardium and papillary muscle in males. Furthermore, soluble collagen increased in most parts of the heart in females, but insoluble collagen increased in all parts of the heart in males. It is concluded that the hydroxyproline content of heart muscle increases in proportion to age and is in inverse proportion to heart weight. The increase occurs in the TCA-soluble and residual fractions, and extends from the endocardium to the papillary muscle. More profound changes in collagen metabolism are observed in males.

Identification of human GATA-2 gene distal IS exon and its expression in hematopoietic stem cell fractions.

Transcription factor GATA-2 is essential for the proper function of hematopoietic stem cells and progenitors. Two first exons/promoters have been found in the mouse GATA-2 gene, and a distal IS promoter shows activity specific to hematopoietic progenitors and neural tissues. To ascertain whether the two-promoter system is also utilized in the human GATA-2 gene, we isolated and analyzed a P1 phage clone containing this gene. The nucleotide sequence of the human GATA-2 gene 5' flanking region was determined over 10 kbp, and a human IS exon was identified in the locus through sequence comparison analysis with that of the mouse GATA-2 IS exon. RNA blotting and reverse-transcribed PCR analyses identified a transcript that starts from the IS exon in human leukemia-derived cell lines. The IS-originated transcript was also identified in CD34-positive bone marrow and cord blood mononuclear cells, which are recognized as clinically important hematopoietic stem cell-enriched fractions. Phylogenic comparison of the human and mouse GATA-2 gene sequences revealed several regions in the locus that exhibit high sequence similarity. These results demonstrate that the GATA-2 gene regulatory machinery is conserved among vertebrates. The fact that the human IS promoter is active in the hematopoietic stem cell/progenitor fraction may be an important clue for the design of a vector system that can specifically express various genes in hematopoietic stem cells and progenitors.

[Histopathological study of resected esophageal cancers between with and without preoperative radiation therapy].

A histopathological study was performed on 53 radiated and 123 non-radiated resected esophageal cancers. The preoperatively radiated (3,000 to 4,000 rads) lesions were usually smaller than the non-treated ones due to the reduction in tumor tissue, and the incidence of infiltrative ulcerated type lesions (Borrmann 3-like) was markedly higher in the former. Histologically, the incidence of moderately-differentiated squamous cell carcinoma (SCC) was lower and that of well-differentiated SCC was higher in the radiated cases, probably due to the high sensitivity of the former type compared with the latter. The incidence of vascular involvement was lower in the radiated cases than the non-treated cases because of the reduction in vessel size due to fibrosis and scar caused by the radiation. However, lymph-nodal metastasis was more frequent in the radiated cases in which the more deeply invaded lesions were included compared with the non-treated cases.

[Study of early gastric carcinoma from the standpoint of size].

1,112 cases of resected early gastric carcinoma without invasion beneath the propria muscularis were investigated for the location, gross findings and histological findings mainly from a standpoint of size. The incidence of intramucosal carcinomas was 92.7% in the size within 1 cm in diameter, 68.9% in 1 to 2.9 cm, 48.8% in 3 to 4.9 cm and 37.6% beyond 5 cm. Although the incidence of poorly-differentiated adenocarcinomas was 37.9% in the cases within 5 cm, the incidence in the cases beyond 5 cm was 50.8% (most of them consisted of signet-ring cell carcinoma). The larger the tumor size, the more the intestinal metaplastic type carcinomas decreased and the signet-ring cell carcinomas increased. It was suggested that the signet-ring cell carcinoma infiltrates in the propria mucosae for a long time compared with the other type carcinomas.

[Clinicopathological study of 65 cases of aberrant pancreas in the stomach].

Sixty-five cases of aberrant pancreas in the gastric wall in 9,650 resected stomach were investigated clinicopathologically. Average age was 44.5 and male to female ratio was 3.5 to 1. Eighty-eight per-cent of the lesions were found in the pylorus and antrum, and the lesions were frequently found in the posterior wall, anterior wall and greater curvature. The size of the lesions varied from 0.7 cm to 4.5 cm in diameter and 89.1% of them were within 3 cm in diameter. The protruded lesions were usually found in the space between submucosa and propria muscularis, and the flat-elevated ones were frequently found in the space between propria muscularis and subserosa. The histological incidence of Heinrich type I, II and III were 38.8%, 52.2% and 9.0%, respectively. There was no case of malignant change in the present series.

[Intestinal metaplasia of the stomach--the significance of single-layer and double-layer metaplasia].

Resected early gastric carcinomas (1,690 cases) and atypical epithelial proliferations (adenomatous lesions, 310 cases) were investigated by the 5 mm-wide step sections. There were two types of intestinal metaplasia; double-layer type, where intestinal metaplasia was in the superficial area with the remaining pyloric or pseudopyloric glands in the deep region of the propria mucosae, and single-layer type, where intestinal metaplasia was usually present in a single layer without remaining deep non-metaplastic glands. These two types of intestinal metaplasia were usually found in the same specimen, however, most of the atypical epithelial proliferations (adenomatous lesions) arose from the area of intestinal metaplasia showing a double-layer type. The mitotic activity was usually found in the transitional zone showing these double-layer intestinal metaplasia. Atypical epithelial cells arose in the transitional zone of the double-layer intestinal metaplasia and spread into the luminal side by budding or replacement of existing epithelial cells. However, the author suspected that the cells in the under area of the transitional zone reproduce non-atypical cells to supplement the cells in the existing pyloric or pseudopyloric glands. On the other hand, atypical epithelial proliferations of single-layer type were rarely found showing a concaved appearance. Some of them may arise from the intestinal metaplasia of single-layer type, where the mitotic region moved towards the lower 1/3 of the glands. It seemed likely that most of the well-differentiated adenocarcinomas arise with intimate relation to these two types of intestinal metaplasia, where the incidence of malignant change of each type has been unknown.

[Study on mucosal carcinomas of the large intestine, polypectomized for the recent 5 years].

Intramucosal carcinomas of the large intestine, polypectomized from 1988 to 1992 were investigated. Out of 111 lesions of intramucosal carcinoma, 106 cases (95.5%) co-existed with adenoma. The incidence of co-existence with adenoma was 0%, 14.4%, 31.5%, 26.1%, 18.9% and 9.0% in the lesions within 4mm, 5-9mm, 10-14mm, 15-19mm, 20-29mm, and over 30mm in diameter, respectively. The incidence of gross findings in the co-existed cases of cancer and adenoma was 0.9%, 15.1%, 32.1%, and 51.9% for IIa, Is, Isp, and Ip types, respectively. Histological incidence of co-existed adenomas was 63.2%, 31.1% and 5.7% for tubular, tubulovillous and villous types. Occupation rate of cancer tissue was within half in the majority of co-existed lesions.

[A resected case of mucin-secreting malignant papillomatosis (villous tumor) of ductus choledocus and left intrahepatic duct].

A resected case of remarkably mucin-secreting malignant papillomatosis (villous tumor) of the ductus choledocus and left intrahepatic duct in a 79 year-old female was reported. In this case, cholecystectomy and choledocho-duodenal anastomosis for the gallbladder stone were performed 14 years ago. Villous or papillary tumors arose in the ductus choledocus and spread into the left intrahepatic duct. Histological examination revealed villous proliferation similar to the villous tumor in the large intestine, in which cancerous and adenomatous changes intermingled showing remarkable mucin secretion. Tumor invasion was limited in the wall without extracanal dissemination, and nodal metastasis was absent. Interestingly, exceeding mucin-secretion was observed as the frog-egg like appearance in the ductus choledocus and as the regurgitation into the stomach by the endoscopic examination. This type carcinomas usually show favorable prognosis compared with the ordinary adenocarcinomas of the bile duct.

[Immunohistochemical study of EGF and EGF-R on the esophageal carcinomas--from a standpoint of depth of invasion].

We performed immunohistochemical stainings for epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R) on 63 resected esophageal carcinomas without preoperative treatment and 12 cases with preoperative radiation to clarify a relationship between positivity and depth of invasion. EGF and EGF-R showed a similar positivity (75% of early cases and 88.9% of advanced ones invaded beyond submucosa). In advanced carcinomas, the positivity in each layer was 75% in the mucosa, 86.7% in the submucosa and muscle layer, and 93.3% in the adventitia. All lesions of nodal metastases were positive for these stainings. Sixty % of cases with preoperative radiation were positive. The degenerated cells showed weak positivity. However, the viable cells showed similar positivity to those of non-treated cases.

[Immunohistochemical study for growth factor and oncogene on atypical epithelium of the stomach].

Almost all atypical epithelial lesions of the stomach consist of atypical cells in the superficial part of the glands and nonatypical cells in the deeper portion of the glands. A transition zone was formed between the superficial atypical gland cells and the deeper nonatypical gland cells. Positive cells were widely demonstrated with immunohistochemical stains for PCNA in the superficial atypical glands and transition zone. The rate of PCNA positivity was 37.7%. However, a small number of positive cells for EGFR (8.5%), c-erbB-2(11.3%), p53(11.3%) and c-K-ras(1.7%) were found in ATP. The incidence of positivity for these factors was low compared with that for carcinomas. The percentages of positive cells for EGFR(1.5%) and c-erbB-2(4.5%) were very low in intestinal metaplasia.

[Histological study of heterotopic pancreas tissue of the stomach--histogenesis and immunohistochemical findings].

Forty resected cases of heterotopic pancreas (15 of Heinrich's type I and 25 of type II) in the stomach were investigated. Acinic cells were more remarkably positive by pancreatic polypeptide and amylase in the cases of type I consisting of acinic cells, ducts and islet cells compared with those in type II consisting of acinic cells and ducts. Staining behavior by insulin, gastrin, glucagon, somatostatin and serotonin was similar to that of normal pancreas. However, the intestinal epithelium containing goblet cells and pyloric gland-like glands consisting of the cells with clear cytoplasm were not infrequently produced from the ducts in the lesions of type II. Muscle fibers with transition to muscularis mucosae were more frequently intermingled in the lesions of type II compared with type I. Acinic cell-differentiation in the mucosa apart from the main lesion was found in several cases of type II. It was suspected that there are two types of histogenesis for the heterotopic pancreas in the stomach; one arises from the fetal immigration of pancreas tissue in the stomach and the other from the immature gastric mucosal penetration into the submucosa with secondary differentiation to the pancreas tissue.

[Clinicopathological study of endometrial-type adenocarcinoma of uterus].

Seventy-one resected cases of endometrial-type adenocarcinoma of uterus were clinicopathologically investigated. As to the gross findings, the incidence of localized type and diffuse type was 47.9% and 52.1%, while that of extroversive type and introversive type was 59.1% and 40.9%, respectively. The incidence of the lesions less than 3 cm in diameter was 36.7% and that beyond 5 cm was 28.1%. The majority (88.7%) of the endometrial-type adenocarcinoma were histologically well-differentiated. Concerning depth of invasion, the incidence of the cases of intramucosal invasion, invasion within 1/3 of muscle layer, invasion between 1/3 and 2/3 of muscle layer, and invasion beyond 2/3 of muscle layer was 19.7%, 46.5%, 16.9%, and 16.9%, respectively. Lymphatic and venous invasion were more frequently found in diffuse, introversive, moderately-differentiated, and deep invasive types than the other types. Lymph nodal metastasis was found in 9.8% of all cases (16.2% in diffuse type, 20.8% in introversive type, 33.3% in moderately-differentiated type and 41.7% in the cases beyond 2/3 of muscle layer).

[Immunohistochemical study of growth factors and oncogenes in gastric carcinomas].

Immunohistochemical staining for EGF, EGFR, c-erbB-2, p53, K-ras and PCNA was performed on the formalin-fixed, paraffin embedded sections of resected gastric carcinomas. A relatively high positive rate was observed for EGFR and c-erbB-2 in the well-differentiated adenocarcinomas and p53 in the poorly-differentiated adenocarcinomas. The positive rate of these factor was higher in the advanced cases than in the early cases, and also in the deep invasive area than the superficial area. According to the PCNA staining, a relatively high positive rate was observed in the well-differentiated adenocarcinomas compared with the early cases of poorly-differentiated adenocarcinomas, but the positive rate was markedly higher in the advanced cases of the latter. Typical signet-ring cell carcinomas showed the lowest positivity rate compared with the other histological types of gastric carcinomas.

[Immunohistochemical study of the lymph-follicles and lymphocytes in the gastric mucosa].

Immunohistochemical stainings according to ABC method (UCHL-1,L-26,MT-1,MB-1,IgG,IgA,IgD,IgM, kappa, lambda, LN-1 and LN-2) for the lymphocytes in the germinal center, mantle zone and infiltrative lymphocytes in the gastric mucosa of 30 cases of chronic gastritis and 10 cases of reactive lymphoreticular hyperplasia (RLH) were performed. Lymphocytes in the germinal center and mantle zone consisted usually of B-cells positively stained by L-26 and MB-1. However, in the interstitially infiltrative cells,T-cells positively stained by UCHL-1 and MT-1 were not infrequently contained. Immunoglobulin stainings revealed marked positivity for IgG,IgA,IgD,IgM, kappa and lambda in the interstitial lymphocytes and plasma cells. As to the RLH, small number of T-cells scattered in the germinal center and surrounding area of lymph follicles, and large number of T-cells were found among the follicles, where B-cells were more infrequently found than in the interstitial area of propria mucosae. Confusion of enlarged germinal centers and monotonous proliferation of lymphocytes among the lymph follicles showing monotonous positivity for the stains of heavy and light chains in the cases of RLH were suggestive of malignant change.

[Immunohistochemical study of PCNA, EGFR, c-erbB-2 and p53 in carcinomas of large intestine].

Immunohistochemical stainings for EGFR, c-erbB-2, p53 and PCNA were performed on 36 and 30 cases of intramucosal and advanced carcinomas of large intestine. Positive rate was 58.3%, 41.6%, 58.3% and 60.6% for EGFR, c-erbB-2, p53 and PCNA in the intramucosal cases, and 66.7%, 50%, 66.7% and 72.6% in the advanced ones, respectively. Relationship between EGFR and c-drbB-2 was more significant in the advanced carcinomas than that in the intramucosal ones. It seemed likely that relationship between p53 and c-erbB-2 was more significant than that between p53 and EGFR. Positive rate of PCNA was of intimate relationship among that of EGFR, c-erbB-2 and p53, and the positive rate increased in the advanced carcinomas.