Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma.

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.

[Clinicopathologic study of 128 cases of T-lymphoblastic lymphoma/leukemia].

OBJECTIVE: To clarify clinical and morphological features and immunophenotype of T lymphoblastic lymphoma/leukaemia (T-LBL/ALL) and to further improve the knowledge and diagnostic accuracy for T-ALL/LBL. METHODS: 128 cases of T-LBL/ALL were analyzed for the clinical features, morphology, immunophenotype and TCR gene rearrangement using routine eosin and haematoxylin stain, immunohistochemistry and polymerase chain reaction combining with the clinical findings. RESULTS: In 128 cases of T-LBL/ALL, there were 94 male and 34 female. The ratio of male/female was 2.8:1. The age of patients ranged from 4 to 88 years, with an average of 27 years and a median of 22 years. Lymph nodes and extranodal areas were involved in 58/128 and 27/128 cases of T-LBL/ALL, respectively. The other 43 cases had involvement of both nodal and extranodal areas. Cervical node and mediastinum were involved in 74 cases and 43 cases, respectively. Diffuse growth pattern of tumor cells was predominant. Nodular growth pattern was seen only in a few cases. Most cases composed of small to medium-sized lymphoblasts, and other 7 cases showed a composition of large lymphoblasts. Tumor cells expressed TdT in 121/128 (94.5%) cases, CD34 in 48/98 (49.0%) cases, CD3 in 78/108 (72.2%) cases, CD7 in 104/108 (96.3%) cases, CD43 in 56/63 (88.9%) cases, CD79a in 5/70 (7.1%) cases, CD10 in 25/76 (32.9%) cases, CD99 in 58/60 (96.7%) cases and Pax-5 in 4/91(4.4%) cases. All of the cases were negative for MPO. A follow up data, ranging from 1 to 53 months, was obtained in 51/128 (39.8%) patients. The over all survival rate was 68.6% and the median survival time was 12 months. Under a similar condition of carrying a positive staining result on CD3 in tumor cells, there was a statistically significant difference between patients in the group of over 30 of age and that with the age ranging from 11 to 30. Patients associating with a CD10 positive staining of tumor cells showed also a shorter survival period. In addition, there were 4 out of 5 cases showing the presence of TCR gene rearrangement. CONCLUSIONS: T-LBL/ALL are aggressive in behavior, associating mainly with enlarged cervical lymph nodes and masses in the mediastinum, occurring predominantly in children and young adults. Although small to medium-sized tumor cells with diffuse pattern were found in most cases, however, large-sized tumor cells and nodular pattern could also be obtained in a few cases. Immunohistochemistry staining particularly adoption of CD7, Pax-5, TdT, CD34 and Ki-67 stainings in combination are helpful of making a diagnosis for T-LBL/ALL. Analysis of TCR gene rearrangement will be helpful for the diagnosis of a few difficult cases.

Ceruloplasmin expression and its role in iron transport in C6 cells.

Ceruloplasmin (CP) is essential for brain iron homeostasis. However, little is known about the effect of iron on CP expression in the brain. Also, the role of CP in brain iron transport has not been well determined. In this study, we investigated the effects of iron on CP expression and the role of CP in iron transport in the C6 rat glioma cells. Our data showed that treatment of the cells with iron (cell iron overload) or iron chelators (cell iron deficiency) did not induce a significant change in the expression of CP mRNA. However, western blotting analysis demonstrated that cell iron overload induced a significant decrease in CP protein content in the cells and that treatment with iron chelators led to a significant increase in CP protein level in the cells. These findings suggest a translational regulation of CP expression by iron in the cells. We also examined the effects of CP on iron transport in the cells. We found that glycosylphosphatidylinositol-anchored CP did not have any impact on iron uptake by normal iron or iron-deficient cells nor on iron release from normal iron or iron-sufficient cells. However, low concentrations of soluble CP (2-8 microg/ml) increased iron uptake by iron-deficient C6 glioma cells, while the same concentrations of CP had no effect on iron uptake by normal iron cells and iron release from normal iron and iron-sufficient cells. The possible reason for the difference between our results in vitro and those obtained from in vivo studies was discussed.

Age-dependent and iron-independent expression of two mRNA isoforms of divalent metal transporter 1 in rat brain.

The DMT1(Nramp2/DCT1) is a newly discovered proton-coupled metal-ion transport protein. The cellular localization and functional characterization of DMT1 suggest that it might play a role in physiological iron transport in the brain. In the study, we evaluated effects of dietary iron and age on iron content and DMT1 expression in four brain regions: cortex, hippocampus, striatum, substantia nigra. Total iron content in all regions was significantly lower in the low-iron diet rats and higher in the high-iron diet rats than that in the control animals, showing that dietary iron treatment for 6-weeks can alter brain iron levels. Contrary to our expectation, there was no significant alternation in DMT1(+IRE) and (-IRE) mRNA expression and protein content in all brain regions examined in spite of the existence of the altered iron levels in these regions after 6-weeks' diet treatment although TfR mRNA expression and protein level were affected significantly, as was expected. The data demonstrates that expression of DMT1(+IRE) and (-IRE) was not regulated by iron in these regions of adult rats. The lack of response of DMT1 to iron status in the brain suggests that the IRE of brain DMT1 mRNA might be not really iron-responsive and that DMT1-mediated iron transport might be not the rate-limiting step in brain iron uptake in adult rats. Our findings also showed that development can significantly affect brain iron and DMT1(+IRE) and (-IRE) expression but the effect varies in different brain regions, indicating a regionally specific regulation in the brain.

Vascular endothelial growth factor and microvascular density in esophageal and gastric carcinomas.

AIM: To observe the relationship between the expression of vascular endothelial growth factor (VEGF), microvascular density (MVD) and the pathological characteristics of esophageal and gastric carcinomas. METHODS: S-P immunohistochemical staining was used to investigate the expression of VEGF in all the specimens. The antibody against factor VIII-related antigen was used to display vascular endothelial cells, and MVD was examined by counting the factor VIII-positive vascular endothelial cells. RESULTS: The positive rates of VEGF expression in esophageal carcinoma and gastric carcinoma were 81.36 % and 67.5 % respectively, and the MVD averaged 41.81+/-8.44 and 34.36+/-9.67 respectively, which were higher than those in benign diseases. The expression of VEGF and MVD were closely correlated with the degree of differentiation, lymphatic metastasis, but not related to depth of cancer invasion. In early stage gastric carcinoma, the rate of expression of VEGF and MVD was lower than that in progressive gastric carcinomas. CONCLUSION: The expression of VEGF is correlated with tumor angiogenesis, and VEGF plays an important role in new blood vessels formation, the expression of VEGF and MVD play an important role in tumor growth and metastasis. MVD and the expression of VEGF may be two important indexes for patients' prognosis.

Effects of development and iron status on ceruloplasmin expression in rat brain.

The increased iron content in the brain of subjects with aceruloplasminemia has implicated ceruloplasmin (CP) as a major factor in the regulation of regional brain iron content. In this study, we investigated the effects of age and iron on CP expression in rat brain. In all four regions, the iron concentrations increased with developmental age. There is a similar trend in age-induced changes in CP mRNA and protein. The CP mRNA and protein levels were both lowest at postnatal day (PND) 7. The expression increased gradually with age, reaching the highest at PND196 in the striatum and substantia nigra, and at PND21 and PND63 in the cortex and hippocampus, respectively. This suggests the existence of an age-dependent pre-transcriptional regulation and a regionally specific effect of age on CP expression in the brain. Although total iron in all four regions was significantly lower in the rats fed with a low-iron diet for 6 weeks and higher in the rats with a high-iron diet than those in the control animals, no significant between-group differences in CP mRNA and protein were found in these animals, except in the substantia nigra where a significant increase in CP protein in high-iron rats was observed, and the reverse in low-iron rats. These findings suggested that the effects of iron on CP expression in the brain may be region-specific, and that regulation of CP expression by iron in the substantia nigra was at the post-transcriptional level.