Correlation between genetic alterations and histopathological subtypes in bronchiolo-alveolar carcinoma and atypical adenomatous hyperplasia of the lung

Bronchiolo-alveolar carcinoma (BAC) is a type of lung adenocarcinoma characterized by growth along the alveolar wall. It is divided into two subtypes: sclerosing BAC (SBAC), which has central fibrosis, and non-sclerosing BAC (NSBAC), which lacks central fibrosis. We compared the genetic alterations in these two types of BAC with those in atypical adenomatous hyperplasia (AAH). There were 39 cases of SBAC, 19 of NSBAC and 20 of AAH. To detect the loss of heterozygosity (LOH) we used the microsatellite markers D3S1234 and D3S1300 on chromosome 3p, IFNA and D9S144 on 9p, and TP53 on 17p. We also used polymerase chain reaction-SSCP analysis and direct sequencing to examine a point mutation of the p53 gene at exons 5-8. At the TP53 locus, the frequencies of LOH showed a statistical rank-difference correlation among AAH, NSBAC and SBAC. On chromosomes 3p and 9p there were no statistical differences of LOH among AAH, NSBAC and SBAC. We detected a significant statistical rank-difference correlation in the p53 mutation among AAH, NSBAC and SBAC. These findings suggest that a process of multistep carcinogenesis from AAH through NSBAC to SBAC might occur in some cases of adenocarcinoma, and LOH of 3p and 9p might be an early event of carcinogenesis, while the p53 mutation might be a later event.     

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin)

We have investigated the effect of the immunomodulator ubenimex (hereafter referred to as bestatin) on the enzymatic degradation of the extracellular matrix by human renal cell carcinoma SN12M cells during the invasive process. The invasion of SN12M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of bestatin in a concentration-dependent manner. However, bestatin did not have any effect on tumor cell adhesion and migration to the extracellular matrices which may be involved in tumor cell invasion. Bestatin inhibited the degradation of type IV collagen by tumor cells, but not by tumor-conditioned medium (TCM), in a concentration-dependent manner. We also found that bestatin inhibited hydrolysing activities towards substrates of aminopeptidases in SN12M cells. Since bestatin was found to inhibit aminopeptidase activity, the inhibition of tumor invasion by bestatin is likely to be associated with its action as an enzyme inhibitor. Bestatin only slightly inhibited tumor cell plasmin activity, which can lead to the conversion of the latent collagenase to the active form, but this slight effect was not significant. The zymography of TCM from SN12M cells showed that the treatment of tumor cells with bestatin resulted in the disappearance of the 68 kDa type IV collagenase-enzyme level (active form) and slight reduction of the 72 kDa type IV collagenase-enzyme level (latent form). These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells, suggesting that the aminopeptidase may partly be associated with the conversion of a latent form of type IV procollagenase to an active form or the secretion of the collagenases from tumor cells.     

Clinical significance of expression of membrane type 1 matrix metalloproteinase and matrix metalloproteinase-2 in human head and neck squamous cell carcinoma

Three different membrane-type matrix metalloproteinases (MT-MMPs) activate in vitro the latent form of matrix metalloproteinase-2 (MMP-2), which is one of the key proteinases in invasion and metastasis of various cancers. We examined the mRNA expression of MT1, 2, and 3-MMPs and MMP-2 in cell lines of head and neck squamous cell carcinoma (HNSCC) and quantitated the relative expression levels in human HNSCC tissues by Northern blotting. The tissue localization of MT1-MMP and MMP-2 was determined by immunohistochemistry and in situ hybridization. Their implications in clinicopathologic factors were statistically evaluated. All cell lines examined consistently expressed MT1-MMP and MMP-2, but not MT2, 3-MMP. In the clinical specimens, there was a significant correlation in coexpression of messenger of RNA (P = .0005) and colocalization by immunohistochemistry (P < .0001) for MT1-MMP and MMP-2. Relative mRNA expression levels of MT1-MMP and MMP-2 in the carcinoma tissues were significantly higher than those of the control tissues (P = .0045 and P = .0122, respectively). Both mRNA expression level and immunopositivity of MT1-MMP significantly correlated with lymph node metastasis (P = .0081 and P = .0193, respectively), which was confirmed by multivariate logistic regression analysis. Immunoreaction of MT1-MMP and its mRNA expression were observed in both carcinoma cells and stromal cells. The localization of MMP-2 closely corresponded to that of MT1-MMP. These observations suggest that MT1-MMP possesses a role as a determinant of lymph node metastasis in HNSCC, and that concurrent expression of MT1-MMP and MMP-2 are involved in progression of HNSCC.     

Stereological analysis of Reinke's crystals in human Leydig cells

Summary Reinke's crystals in human Leydig cells were analyzed stereologically to assess their functional role. Testicular tissues were taken from seven older men (57–82 years old) with prostatic carcinoma and also from seven younger men (26–38 y.o.) complaining of male infertility. Sections 0.5 m thick, stained with toluidine blue or Heidenhein's iron-hematoxylin were examined by a point-counting method and with a Particle Measurement Computor System (MC). When the patients were grouped by age, the mean crystal volume, the number of crystals per cell, the volume of crystals per cell and the volume ratio of crystals to cell were significantly larger in the older age group than in the younger age group. In particular, the latter three variables correlated well with the age of subjects, with correlation coefficients of r=0.66–0.85. On the other hand, none of these variables had any correlation with the concentration of plasma testosterone. These results indicate that Reinke's crystals can be considered as degenerative products in cell life but not as facultative constituents for testosterone production.Supported in part by a grant from Medical Foundation of Ehime, Japan     

Stoichiometric salt-linkage formation of trimethylammonium glycol chitosan iodide with a carboxylic type ion exchanger

The adsorption of trimethylammonium glycol chitosan iodide (TGCI) on Dowex CCR-2 resin was investigated at different pH values and resin weights. The concentration of Na⁺ or Ca²⁺ ions, released by the ion exchange reaction with TGCI, was measured by inductively coupled plasma spectroscopy. It was found that the amount of the released ions is comparable to that of the absorbed TGCI. This indicates that the salt-linkage formation of carboxylate ions of the exchanger with ⊕N(CH₃)₃-groups in TGCI follows a stoichiometric relationship. This result is discussed in connection with the form of the polymer layer adsorbed on the solid surface.     

Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG,interleukin-1 and tumour necrosis factor-alpha

Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1alpha (10 ng/ml) and tumour necrosis factor-alpha (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary.     

Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis

Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM. Received: August 30, 2001 / Accepted: November 2, 2001     

The corrosion resistance of pure titanium in organic acids

The purpose of this study was to assess the corrosive properties of titanium at various pH values. Cast pure titanium specimens were immersed in 128 mmol/l of lactic and formic acids at pH 1.0-8.5 for 3 weeks at 37 degrees C. The solubility, color, weight and chemical binding state of specimens were observed. Titanium dissolved in all lactic acid. The amount of dissolved titanium tended to decrease with a higher pH. In formic acid, the amount of dissolved titanium at pH 1.0 was larger than that in lactic acid at the same pH, but less than the detectable limit at pH 4.0 or higher. Significant discoloration was macroscopically observed only in formic acid at pH 2.5 and 4.0. The weight of the titanium samples immersed in lactic acid all decreased, but it was not affected by pH. In formic acid, the weight decreased at pH 1.0 and increased at pH 2.5-5.5. Thickening of the TiO2 corresponding to that showing discoloration was observed in the superficial oxide film of the titanium samples. Our results show that the corrosive properties of titanium are markedly dependent on pH in formic acid, and relatively less dependent on pH in lactic acid in which titanium is dissolvable at pH 1.0-8.5.     

A histological evaluation for guided bone regeneration induced by a collagenous membrane

This study was designed to evaluate the histological changes during ossification and cellular events including osteogenic differentiation responding to collagenous bioresorbable membranes utilized for GBR. Standardized artificial bony defects were prepared at rat maxillae, and covered with a collagenous bioresorbable membrane. These animals were sacrificed at 1, 2, 3 and 4 weeks after the GBR-operation. The paraffin sections were subject to tartrate resistant acid phosphatase (TRAP) enzyme histochemistry and immunohistochemistry for alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC). In the first week of the experimental group, woven bone with ALP-positive osteoblasts occupied the lower half of the cavity. The collagenous membrane included numerous ALP-negative cells and OP-immunoreactive extracellular matrices. At 2 weeks, the ALP-, OP- and OC-immunoreactivity came to be recognizable in the region of collagenous membrane. Since ALP-negative soft tissue separated the collagenous membrane and the new bone originating from the cavity bottom, the collagenous membrane appeared to induce osteogenesis in situ. At 3 weeks, numerous collagen fibers of the membrane were embedded in the adjacent bone matrix. At 4 weeks, the membrane-associated and the cavity-derived bones had completely integrated, showing the same height of the periosteal ridge as the surrounding alveolar bones. The collagen fibers of a GBR-membrane appear to participate in osteogenic differentiation.