Measurement and visualization of cell membrane surface charge in fixed cultured cells related with cell morphology.

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.

Adipophilin expression in cutaneous malignant melanoma is associated with high proliferation and poor clinical prognosis.

Adipophilin (ADP) is a primary protein component of lipid droplets (LDs). For more than half a century, certain types of cancer cells have been known to contain LDs in their cytoplasm. However, the pathological significance of ADP or LDs in cancer remains unclear. In the present study, we investigated the association between ADP and other pathological characteristics in cutaneous malignant melanomas to clarify the role of ADP in melanoma cells. We immunostained whole paraffin sections of primary cutaneous melanomas obtained from 90 cases for ADP, after which we analyzed the correlation between ADP immunohistochemistry (IHC) and patient survival data. We also studied the relationship between the ADP IHC score and in situ hybridization (ISH) score of ADP mRNA, and the Ki67-labeling index (Ki67-LI) by using tissue microarrays consisting of 74 primary cutaneous malignant melanomas, 19 metastasizing melanomas, and 29 melanocytic nevi. Finally, we analyzed the relationship between ADP expression and cell proliferation in cutaneous melanoma cell lines. We found that high ADP expression was associated with poor metastasis-free survival, disease-specific survival, and overall survival rates of patients with cutaneous melanomas (P < 0.05). By linear regression analysis, ADP IHC was correlated with increasing ADP mRNA ISH H-scores and Ki67-LI scores in melanocytic lesions (P < 0.01). ADP IHC and ADP ISH H-scores and Ki67-LI scores were greater in pT3-4 melanomas than in pT1-2 melanomas. In cell-based assays, cells with increased ADP expression showed higher proliferation rates compared with those of low-ADP cells. Thus, ADP expression in malignant melanoma was significantly associated with high cell proliferation and poor clinical prognosis. Our results thus indicate a significant association between ADP and melanoma progression, and we propose that ADP may be a novel marker of aggressive cutaneous melanoma with a lipogenic phenotype.

HER2 is frequently overexpressed in hepatoid adenocarcinoma and gastric carcinoma with enteroblastic differentiation: a comparison of 35 cases to 334 gastric carcinomas of other histological types.

AIMS: α-Fetoprotein (AFP)-producing gastric carcinoma (AFPGC) is one of the most aggressive GC subtypes. Frequent expression of human epidermal growth factor receptor 2 (HER2) has previously been reported in hepatoid adenocarcinoma (HAC), a major histological subtype of AFPGC originating from common-type GC (CGC). However, HER2 expression levels in other AFPGC histological subtypes are unknown. In this study, we analysed HER2 expression in GCs with primitive phenotypes in addition to HAC. METHODS: HER2 expression was evaluated in representative complete sections from 16 HACs, 19 GCs with enteroblastic differentiation (GCEDs) and 334 GCs of other histological types as controls. The Ruschoff/Hofmann method was used to score HER2 immunohistochemistry. Samples with a HER2 score of 2+ were further assessed using fluorescence in situ hybridisation. Oncofetal protein (OFP) expression in HAC and GCED was tested via immunohistochemical staining for AFP, glypican 3 and Sal-like protein 4. RESULTS: Thirty of 35 HAC/GCED cases comprised more than two histological patterns. The HER2 positivity rates of each histological component in the HACs/GCEDs were 25.0% for HAC (n=16), 34.6% for GCED (n=26) and 48.1% for CGC (n=27), which were higher than those of the control group (13.8%). Additionally, the majority of CGC components in HACs/GCEDs were positive for OFP (88.9%). CONCLUSIONS: HER2 is frequently overexpressed not only in HAC but also in GCED and CGC components of HACs/GCEDs, which suggests an association between HER2 and OFP expression. Moreover, our findings suggest that HER2-positive CGC has a higher risk of progression to HAC/GCED than HER2-negative GC.

N-substituted hydroxyureas as urease inhibitors.

In order to seek a urease inhibitor more potent than hydroxyurea (1), its alkyl- or phenyl-substituted derivatives were synthesized and evaluated for their effect on the jack bean urease. Of 16 compounds tested, m-methyl- (10) and m-methoxy-phenyl substituted hydroxyurea (13) showed the most potent inhibitory activities against the enzyme.