論文

© The Author(s) 2020. Published by Oxford University Press. Although some previous studies have examined epigenomic alterations in lung adenocarcinomas, correlations between epigenomic events and genomic driver mutations have not been fully elucidated. Single-CpG resolution genome-wide DNA methylation analysis with the Infinium HumanMethylation27 BeadChip was performed using 162 paired samples of adjacent normal lung tissue (N) and the corresponding tumorous tissue (T) from patients with lung adenocarcinomas. Correlations between DNA methylation data on the one hand and clinicopathological parameters and genomic driver mutations, i.e. mutations of EGFR, KRAS, BRAF and HER2 and fusions involving ALK, RET and ROS1, were examined. DNA methylation levels in 12 629 probes from N samples were significantly correlated with recurrence-free survival. Principal component analysis revealed that distinct DNA methylation profiles at the precancerous N stage tended not to induce specific genomic driver aberrations. Most of the genes showing significant DNA methylation alterations during transition from N to T were shared by two or more driver aberration groups. After small interfering RNA knockdown of ZNF132, which showed DNA hypermethylation only in the pan-negative group and was correlated with vascular invasion, the proliferation, apoptosis and migration of cancer cell lines were examined. ZNF132 knockdown led to increased cell migration ability, rather than increased cell growth or reduced apoptosis. We concluded that DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of 'pan-negative' lung adenocarcinomas. In addition, DNA methylation alterations at the precancerous stage may determine tumor aggressiveness, and such alterations that accumulate after driver mutation may additionally modify clinicopathological features through alterations of gene expression.

p21(WAF1/CIP1) is a universal cyclin-dependent kinase (cdk) inhibitor, the expression of which is regulated by p53-dependent and p53-independent pathways. We examined p21(WAF1/CIP1) expression in and p53 status of 21 primary hepatocellular carcinomas (HCCs) by reverse-transcriptase polymerase chain reaction (RT-PCR) and by PCR single-strand conformation polymorphism (PCR-SSCP) analysis. p21(WAF1/CIP1) messenger RNA expression was reduced markedly in 8 of 21 HCCs (38.1%) and 5 of these 8 HCCs bore p53 mutations. The relative p21(WAF1/CIP1) messenger RNA expression value of HCCs with p53 mutations (.73 ± .13 U, n = 6) was significantly lower than that of HCCs with wild-type p53 (1.00 ± 0.21 U, n = 14; P < .01). The p21(WAF1/CIP1) expression levels in cancerous tissues (.73 ± .13 U) were significantly reduced in comparison with those in noncancerous tissues (.97 ± .13 U) (P < .01) in the 6 cases with p53 mutations. These data indicate that p21(WAF1/CIP1) expression in HCCs is predominantly regulated by dependence on p53 and that reduced p21(WAF1/CIP1) expression may participate in hepatocarcinogenesis.

We report a case of primary low-grade B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type of the urinary bladder. The patient, a 77-year-old woman, presented with a sense of urinary retention. An intravenous pyelogram and cystoscopy revealed a wide-based submucosal mass measuring 3 cm in the left wall of the urinary bladder. Histological findings of the tissue obtained by transurethral resection (TUR) showed a dense, monomorphic atypical lymphoid (centrocyte-like) infiltrate with reactive lymph follicles in the subepithelial tissue. Monocytoid and plasmacytoid features were readily evident in a population of these cells. Lymphoepithelial lesions involving the urothelium were also noticed in some areas. These features were strongly suggestive of primary low-grade lymphoma of the MALT type. The diagnosis was confirmed by immunohistochemical and flow cytometric studies, both of which showed a clear immunoglobulin restriction to lambda light chain and also by polymerase chain reaction-based assay using a formalin-fixed paraffin-embedded TUR tissue sample, which showed a clonal Ig heavy-chain gene rearrangement. Clinical staging procedures revealed that the tumor was localized in the urinary bladder. The patient has not received chemotherapy and is alive and well with no evidence of recurrence, 3 years after TUR. This case demonstrates that these ancillary tests are worth performing for confirmation of B-cell clonality in TUR tissue samples showing dense B-lymphocytic infiltration. © 1999 Foundation for Promotion of Cancer Research.

Purpose: The aim of the present study was to investigate the biological and molecular basis of intraepithelial spreading (IES) of carcinomas in situ (CIS) of the urinary bladder, which were considered to be precursors of nodular invasive carcinomas. Materials and Methods: The propensity for IES of human transitional carcinoma cells was examined by inoculation into murine renal pelvis and urinary bladder, and the biological character of the cells with a high propensity for IES was explored in vitro. Results: Three of 6 cell lines exhibited a high propensity for IES. When cultured on laminin, these IES cells scattered, whereas 3 non-IES cells and 2 immortalized transitional epithelial cells did not. IES cells showed strong adhesiveness, haptotaxis and enhanced migration on laminin compared with both non-IES and immortalized transitional epithelial cells. In IES cells, expression of the integrin β4 subunit was markedly reduced and the integrin α6β1 complex was predominant compared with the integrin α6β4 complex. Transfection of IES cells with integrin β4 subunit cDNA inhibited their ability to migrate on laminin and their propensity for IES. In addition, expression of the integrin β4 subunit was reduced in surgically resected specimens of CIS of the urinary bladder. Conclusion: The results indicate that a reduction in integrin β4 plays a role in IES of CIS of the urinary bladder through enhanced migration on laminin.

To assess the time course of alterations in pulmonary endothelial vasodilator functions during pathological development of pulmonary hypertension (PH) and right ventricular hypertrophy (RVH), we examined changes in serotonin (5-HT) removal rates and the production of prostacyclin (PGI2) and nitric oxide (NO) in isolated rat lungs harvested at various times after single exposure to monocrotaline (MCT). We assessed the generation of vasodilator substances under conditions of both the absence and the presence of 5-HT in lungs perfused with blood-free solution. Major findings included: (i) remodeling of the pulmonary vasculature associated with RVH evident 14 days after MCT injection; (ii) the capacity for 5-HT removal was suppressed at day 1 and 7 but had been restored by day 14 after MCT exposure; (iii) basal PGI2 production in the absence of 5-HT was augmented at day 1 but had returned to control levels in lungs harvested 7 or 14 days postinjection of MCT; (iv) PGI2 production evoked by 5-HT was suppressed in MCT lungs obtained at all time points examined; (v) basal NO production was suppressed at day 1 but enhanced at day 7 and 14 in MCT lungs; (vi) NO production elicited by 5-HT stimulation in 1-day-MCT lungs was obviously suppressed while that in 7- and 14-day-MCT lungs had been restored to the control level. These findings suggest that transitional changes in endothelial functions including 5-HT removal and production of vasodilators in MCT lungs do not follow the same time course.

Objective: The aim was to clarify the role of serotonin (5-hydroxytryptamine, 5-HT) in monocrotaline induced pulmonary hypertension. Methods: Plasma 5-HT levels, pulmonary capillary platelet count, and vascular responsiveness to 5-HT were evaluated in the model. The effects of the selective 5-HT2 receptor antagonist, DV- 7028, on the development of pulmonary hypertension were also investigated. Results: Plasma 5-HT was raised 12 h to 3 d after monocrotaline administration (60 mg·kg-1), coinciding with accumulation of platelets in the pulmonary circulation. Isolated pulmonary arteries showed hyperreactivity to 5-HT at 14 and 21 d after monocrotaline. Administration of DV-7028 (20 mg·kg-1·d-1) attenuated the increase in pulmonary arterial pressure, right ventricular hypertrophy, and medial thickening of the pulmonary arteries. Conclusions: The present study suggests that 5-HT released from platelets contributes to the initiation and progression of monocrotaline induced pulmonary hypertension.Cardiovascular Research 1993;27:1619-1623.

Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including α- and β-catenins. While α-catenin has been demonstrated to be crucial for cadherin (unction, the role of β-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of β-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the β-catenin gene. On the other hand, these cells expressed E-cadherin, α-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated β-catenin but not with α-catenin, and antibodies against β-catenin did not copurify α-catenin. However, the recombinant fusion protein containing wild-type β-catenin precipitated α-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with α-catenin, and that this defect results from the mutation in β-catenin. Thus, it is most likely that the association between E-cadherin and α-catenin is mediated by β-catenin, and that this process is blocked by NH2-terminal deletion in β-catenin. These findings indicate that genetic abnormality of β-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers. © 1994, American Association for Cancer Research. All rights reserved.

Invasion and metastasis are later but critically important steps in multistage carcinogenesis. The inactivation of E (epithelial)-cadherin, which is a Ca2+-dependent cell-cell adhesion molecule, may trigger the release of cancer cells from the cancer nests. In fact, the invasion suppressing function of E-cadherin has been shown in in vitro studies. Furthermore, E-cadherin expression was strong in well differentiated carcinomas, whereas generally reduced in undifferentiated ones showing the strong invasive property. In spite of the above general rule, we found that a sizable proportion of undifferentiated gastric adenocarcinomas showing scattered histological growth pattern had definite E-cadherin expression. Therefore, human carcinoma cell lines lacking tight cell-cell adhesion were examined to clarify if alterations in catenins, which are cytoplasmic proteins interacting with cadherins, and/or structural abnormalities of normally expressed E-cadherin caused the dysfunction of the cell-cell adhesion system. Homologous deletion of part of the α-catenin gene with markedly reduced expression was observed in a human lung cancer cell line, PC9. mRNA sequence abnormalities of E-cadherin were detected in two human gastric cancer cell lines, MKN 45 and KATO-III. The wild-type allele of E-cadherin locus was lost in the two cell lines, indicating that E-cadherin gene was inactivated by a combination of loss of gene and mutation, similar to the mechanism which inactivated tumor suppressor genes. These findings suggested that varying genetic abnormalities of cell adhesion molecules could participate in multistage carcinogenesis, especially in the processes of invasion and metastasis.

The cell-surface adhesion molecule α6β4-integrin is a receptor for laminins and a component of hemidesmosomes. β4-Integrin expression is restricted to proliferating basal keratinocytes in the epidermis and is suppressed when differentiation commences. Altered β4-integrin expression levels correlate significantly with the aggressive behavior of cancers. In order to clarify the mechanisms that regulate transcription of the β4- integrin gene, we cloned its 5'-flanking region. This 5'-flanking region was found to have a high G + C content and not to contain either TATA or CAAT boxes. Nested delimitation and reporter analyses mapped a basal promoter to nucleotides -106 to +105, surrounding the most proximal transcription initiation site. Gel retardation and mutational analyses revealed that cooperation between AP1 and Ets, interacting with other factors, mediated the promoter activity. In addition to the promoter element, enhancer activity was found in the first intron (+1905/+3933) and in a sequence upstream of the promoter region (-414/-107). These findings should facilitate our understanding of the regulation of β4-integrin gene expression in processes such as cell growth and differentiation, apoptosis, and cancer development and metastasis.