MicroRNA-mediated upregulation of integrin-linked kinase promotes Src-induced tumor progression.

The tyrosine kinase c-Src is upregulated in various human cancers; however, the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. Here we show that downregulation of microRNA (miR)-542-3p is tightly associated with tumor progression via c-Src-related oncogenic pathways. In c-Src-transformed fibroblasts and human cancer cells that overexpress c-Src, miR-542-3p is substantially downregulated, and the ectopic expression of miR-542-3p suppresses tumor growth. We identified the integrin-linked kinase (ILK) as a conserved target of miR-542-3p. ILK upregulation promotes cell adhesion and invasion by activating the integrin-focal adhesion kinase (FAK)/c-Src pathway, and can also contribute to tumor growth via the AKT and glycogen synthase kinase 3ß pathways. MiR-542-3p expression is downregulated by the activation of c-Src-related signaling molecules, including epidermal growth factor receptor, K-Ras and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase. In human colon cancer tissues, downregulation of miR-542-3p is significantly correlated with the upregulation of c-Src and ILK. Our results suggest that the novel c-Src-miR-542-3p-ILK-FAK circuit plays a crucial role in controlling tumor progression.

MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.

The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor growth remain unclear. Here we examined the involvement of microRNAs in the c-Src-mediated tumor growth. Microarray profiling revealed that c-Src activation downregulates a limited set of microRNAs, including miR-99a, which targets oncogenic mammalian target of rapamycin (mTOR) and fibroblast growth factor receptor 3 (FGFR3). Re-expression of miR-99a suppressed tumor growth of c-Src-transformed cells, and this effect was restored by the overexpression of mTOR. The downregulation of miR-99a was also observed in epidermal growth factor- and Ras-transformed cells, and it was suppressed by inhibiting the mitogen-activated protein kinase (MAPK) pathway. Furthermore, miR-99a downregulation is associated with mTOR/FGFR3 upregulation in various human lung cancer cells/tissues. The tumorigenicity of these cells was suppressed by the introduction of miR-99a. These findings suggest that the miR-99a-mTOR/FGFR3 pathway is crucial for controlling tumor growth in a wide range of human cancers that harbor upregulation of the Src-related oncogenic pathways.

Diffuse large B-cell lymphoma showing an interfollicular pattern of proliferation: a study of the Osaka Lymphoma Study Group.

AIMS: Diffuse large B-cell lymphoma (DLBCL) usually proliferates effacing lymph follicles. In occasional cases, tumour cells show an interfollicular pattern of proliferation preserving lymph follicles. The aim was to analyse clinicopathological findings in DLBCL showing an interfollicular pattern of proliferation to determine whether this type of lymphoma is a distinct entity of DLBCL. METHODS AND RESULTS: Clinicopathological findings in 12 cases of DLBCL showing an interfollicular pattern of proliferation [interfollicular group (IF)] were examined and compared with those in 30 cases of DLBCL with ordinary morphology [control group (CG)]. IF showed a significantly lower lactate dehydrogenase level and International Prognostic Index scores than CG (P = 0.023 and P < 0.01, respectively). The frequency of localized disease, clinical stage 1 and 2, in IF was higher than that in CG (P = 0.016). A morphologically polymorphous pattern of proliferation was found in seven of 12 cases (58.3%) in IF, which was higher than that in CG, five (16.7%) of 30 cases (P < 0.01). Clonality analysis with the polymerase chain reaction method revealed that all 11 IF cases examined showed a monoclonal pattern. Immunohistochemically, the majority (11 of 12) of IF cases showed a non-germinal centre B-cell phenotype and the frequency was higher than that in CG (P = 0.021). CONCLUSION: Diffuse large B-cell lymphoma with an interfollicular pattern of proliferation shows distinct clinical and pathological findings from ordinary DLBCL.

Cell origin of pyothorax-associated lymphoma: a lymphoma strongly associated with Epstein-Barr virus infection.

Pyothorax-associated lymphoma (PAL) is an Epstein-Barr virus (EBV)-associated B cell lymphoma developing in the pleural cavity affected by chronic pyothorax. To clarify the cell origin of PAL, the expression of immunoglobulin heavy (IgH) and light chains in relation to somatic hypermutations (SHMs) of rearranged Ig heavy- and light-chain variable (IgV(H), IgV(L)) genes was examined using cell lines as well as clinical samples. SHMs without ongoing mutations of the IgV(H) gene were found in all PAL cell lines and clinical samples available for sequencing, indicating PAL to be derived from B cells at the postgerminal center (GC) stage of the differentiation process. They could be subdivided into post-GC cells with potentially productive IgV(H) genotypes (Group 1) and with sterile IgV(H) genotypes (Group 2). IgH expression was abrogated in Group 2 as expected and also in two cell lines in Group 1. DNA demethylation experiments with 5-aza-dC induced expression of IgH mRNA and protein in these cell lines. Most PAL cells were derived from crippled post-GC cells, which usually could not survive. Transformation of such B cells through EBV infection might provide a basis for the development of PAL with additional genetic changes.

Decreased number of mast cells infiltrating into needle biopsy specimens leads to a better prognosis of prostate cancer.

Mast cell infiltration is often observed around human tumours. Inflammatory cells such as macrophages, neutrophils and mast cells infiltrating around tumours are known to contribute to tumour growth; however, the clinical significance of mast cell invasion in prostate cancer (PCa) has not been investigated. Mast cell infiltration was evaluated in 104 patients (age range, 45-88 years; median, 72 years), who underwent needle biopsy of the prostate and were confirmed to have PCa. Needle biopsy specimens of prostate were sliced into 5-microm-thick sections and immunostained for mast cells with monoclonal antibody against mast cell-specific tryptase. Mast cells were counted systematically under a microscope (x 400 magnification), and the relations between mast cell numbers and clinicopathologic findings were evaluated. The mast cell count was evaluated for prognostic value by multivariate analysis. Mast cells were immunostained around the cancer foci. The median number of mast cells in each case was 16. The mast cell count was higher around cancer foci in patients with higher Gleason scores than in those with low Gleason scores. The mast cell number correlated well with clinical stage (P<0.001). Prostate-specific antigen-free survival of patients with higher mast cell counts was better than that in patients with lower mast cell counts (P<0.001). Multivariate analysis revealed that mast cell count was a significant prognostic factor (P<0.005). The number of mast cells infiltrating around cancer foci in prostate biopsy specimens can be a significant prognostic factor of PCa.

Unicentric and multicentric Castleman's disease.

Castleman's disease (CD) appears at ubiquitous lymph nodes. To date, detection of the lesion focus for CD has mainly been carried out by physical examination and radiological findings, such as X-ray analysis, CT and MRI. 18F-FDG PET visualizes the active focus of glucose metabolism and the clinical value has been investigated for many different tumours. Previous studies of 18F-FDG PET for CD have only reported four cases of unicentric CD and no cases of multicentric CD. In this paper, we report two cases of CD, one with unicentric CD and one with multicentric CD. We demonstrate that the use of 18F-FDG PET for the detection and monitoring of patients with CD, especially multicentric CD, would be effective.

Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells.

CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38- cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.

Epigenetic regulation of the expression of the novel stem cell marker CDCP1 in cancer cells.

CDCP1 is a novel stem cell marker that is expressed in several types of cancer. The mechanisms by which CDCP1 expression is regulated, and the clinical implications of this marker, have not been clarified. In this report, we examine the epigenetic regulation of CDCP1 expression in cell lines and clinical samples from patients with breast cancer. Many CpG sequences were localized around the transcription initiation site of CDCP1. These CpG motifs were found to be poorly methylated in cell lines with high levels of CDCP1 expression and heavily methylated in cell lines with low levels of CDCP1 expression. The in vitro methylation of CpG sites decreased CDCP1 promoter activity, and the addition of a demethylating reagent restored activity. In 25 breast cancer samples, an inverse correlation was noted between the CDCP1 expression level and the proportion of methylated to non-methylated CpG sites. Tumours with high-level CDCP1 expression showed higher levels of proliferation, as revealed by immunohistochemical detection of the MIB-1 antigen, than tumours with low-level CDCP1 expression. These findings indicate that the expression of CDCP1 is regulated by methylation of its promoter region in tumours. CDCP1 expression may prove to be useful in the further characterization of cancers.

Increased expression of renin in chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is the main cause of renal transplant failure in the first decade posttransplant. The precise pathogenetic mechanism for CAN is not completely understood. A possible role of renin-angiotensin system for CAN has been suggested through clinical observations that angiotensin-converting enzyme inhibition and angiotensin II receptor blockers prevent CAN. METHODS: Distribution of renin-positive cells in allograft biopsy specimens was examined immunohistochemically in 23 renal transplant recipients diagnosed with CAN Biopsy specimens obtained from seven recipients with stable renal function were examined as controls. Histologic evaluation was performed based on the Banff 97 classification. RESULTS: Renin-positive cells were found in the juxtaglomerular apparatus (JGA) adjoining the afferent arterioles in both groups. When the number of renin-positive cells in JGA was defined as a renin index, it was significantly higher in the CAN than the control group (P = .007). There was no significant difference in age, interval between transplantation and biopsy, and blood pressure between groups. Only a significantly higher serum creatinine was found in the CAN group. CONCLUSIONS: The increased renin-positive cells in JGA suggest a significant role of the intrarenal renin-angiotensin system activation in the development of CAN.

Expression of inhibin alpha, glial cell line-derived neurotrophic factor and stem cell factor in Sertoli cell-only syndrome: relation to successful sperm retrieval by microdissection testicular sperm extraction.

BACKGROUND: Microdissection testicular sperm extraction (TESE) has provided new hope for successful sperm retrieval to patients with Sertoli cell-only syndrome (SCO). We determined expression of the inhibin alpha subunit, glial cell line-derived neurotrophic factor (GDNF) and stem cell factor (SCF) in Sertoli cells obtained from patients with SCO immunohistochemically and compared expression rates with rates of microdissection TESE sperm retrieval. METHODS: Testicular biopsy specimens were obtained from 52 men with non-obstructive azoospermia who underwent microdissection TESE and were diagnosed with SCO by histological analysis. RESULTS: All specimens showed intense staining for the inhibin alpha subunit. Moderate or intense staining for GDNF was observed in 65.8% of specimens. All but one showed moderate or intense staining for SCF. Among specimens negative for GDNF, the sperm retrieval rate was significantly higher (100%) for specimens with intense staining for SCF than for specimens with no or moderate staining (30.7%) (P<0.05) for SCF. CONCLUSION: GDNF expression differs among patients with SCO. The sperm retrieval rate was high in cases of no staining for GDNF and intense staining for SCF.

Expression level of valosin-containing protein (VCP) as a prognostic marker for gingival squamous cell carcinoma.

BACKGROUND: Valosin-containing protein (VCP) is associated with anti-apoptotic function and metastasis via activation of the nuclear factor-kappaB signaling pathway. In the present study, association of VCP expression with prognosis of gingival squamous cell carcinoma (GSCC) was examined. PATIENTS AND METHODS: VCP expression in 74 patients with GSCC (34 males and 40 females) with ages ranging from 42 to 85 (median 66) years was evaluated by immunohistochemistry, in which staining intensity in tumor cells was categorized as either weaker (level 1) or equal to/stronger (level 2) than that in the endothelial cells. RESULTS: Twenty-four (32.4%) cases showed level 1 and 50 (67.6%) level 2 VCP expression. Patients with level 1 GSCC showed a significantly better 5-year survival rate than those with level 2 GSCC (5-year overall survival: 100% versus 84.9%, P < 0.05). Multivariate analysis revealed VCP expression level, lymph node metastasis and pT(TNM) to be independent factors for overall survival. Patients with GSCC at stages I and II showed favorable prognosis regardless of VCP expression status, whereas at stages III and IV, patients with level 1 VCP expression showed better survival rates than those with level 2 expression. CONCLUSION: Prognostic significance of VCP expression level in GSCC was demonstrated.

Risk factors for graft loss in patients with recurrent IGA nephropathy after renal transplantation.

BACKGROUND: The recurrence rate of IgA nephropathy (IgAN) in transplanted kidneys has been reported to be >50%. Although recurrent IgAN has a benign clinical course, recent data suggest that it leads to graft loss in a substantial number of patients. METHODS: We performed a retrospective single-center analysis of 34 renal transplant recipients, with biopsy-proven IgAN as the cause of end-stage renal failure. RESULTS: Renal allograft biopsies were performed in 30 patients, of whom 24 did and 6 did not have biopsy-confirmed recurrent transplant IgAN. Recurrent transplant IgAN was more often detected in men and at later timepoints after post-transplantation. Four patients with recurrent transplant IgAN progressed to graft failure. Progression to graft failure was associated with worsened renal function, higher systolic blood pressure, and the lack of presenation of angiotensin-converting enzyme inhibitors (ACEs) at the time of allograft biopsy. Immunologic factors such as frequency of acute rejection, HLA typing, and immunosuppression did not show a relation to recurrence or graft loss. CONCLUSIONS: Recurrent transplant IgAN increased with long-term graft survival and risk factors for graft loss due to recurrent IgAN were similar to those among IgAN in native kidneys.

Characterization of low-intensity lesions in the peripheral zone of prostate on pre-biopsy endorectal coil MR imaging.

The aim of this study was to determine which morphological features of low-intensity lesions in the peripheral zone of the prostate are predictable of prostate cancer on pre-biopsy T2-weighted integrated endorectal phased-array MR images. The MR examinations were performed in 69 consecutive patients with elevated level of prostate-specific antigen (>4 ng/ml) and/or a positive digital rectal examination before transperineal 12-site biopsy. Two radiologists evaluated presence of lesions, their morphological features, and possibility of malignancy in divided into four sections of the peripheral zone. Imaging analysis findings were compared with biopsy results. Discriminative features were selected by stepwise logistic regression. Descriptive statistics and receiver operating characteristics (ROC) curves were also calculated. Sixty-eight benign lesions and 23 malignant lesions were found. Wedge shape and diffuse extensions without mass effect were significantly associated with benignity ( P=0.0105 and 0.002, respectively). Lesion size was significantly associated with malignancy ( P=0.0001). For evaluating probability of malignancy for lesions, regression model showed a comparable accuracy with the total impression for the readers in ROC analysis (Az 0.9095 vs 0.9266, respectively). Wedge shape, diffuse extension without mass effect, and size are the morphological features of low-intensity lesions in the peripheral zone on pre-biopsy T2-weighted MR images that give the best prediction of malignancy.

Potential mechanism for the effects of dexamethasone on growth of androgen-independent prostate cancer.

BACKGROUND: Dexamethasone, a synthetic glucocorticoid, has clinical benefit in patients with hormone-refractory prostate cancer (HRPC), but the mechanisms responsible for its effects are unknown. The nuclear factor-kappaB (NF-kappaB)-dependent cytokine interleukin (IL) 6 (IL-6) is thought to stimulate growth of HRPC. Because dexamethasone interferes with NF-kappaB activation, we determined whether dexamethasone inhibits prostate cancer growth by working through the glucocorticoid receptor (GR) to interfere with NF-kappaB-IL-6 pathway. METHODS: Three human prostate cancer cell lines (DU145, PC-3, and LNCaP) were assessed for GR expression and responsiveness to dexamethasone. Levels of GR, NF-kappaB, and the cytoplasmic NF-kappB inhibitor IkappaBalpha were determined by western blotting and of IL-6 by enzyme immunoassay. The subcellular localization of NF-kappaB was analyzed by immunofluorescence. The effects of dexamethasone (thrice weekly injections of 1 microg/mouse) on DU145 xenografts in nude and severe combined immunodeficient (SCID) mice were evaluated. GR expression in human prostate cancers was assessed by immunohistochemistry. All statistical tests were two-sided. RESULTS: Dexamethasone dose dependently decreased GR levels and inhibited the growth of DU145 and PC-3 but not LNCaP cells (DU145 cells, P< .001; PC-3 cells, P = .009). Dexamethasone increased IkappaBalpha protein levels and the cytosolic accumulation of NF-kappaB in DU145 cells and decreased secreted IL-6 levels to 37 pg/mL (95% confidence interval [CI] = 33 pg/mL to 41 pg/mL), compared with 164 pg/mL (95% CI = 162 pg/mL to 166 pg/mL) secreted by ethanol-treated control cells. Dexamethasone inhibited the growth of DU145 xenografts in nude (P = .006) and SCID (P = .026) mice without affecting GR levels. Eight of 16 human prostate cancers expressed GR at high levels (>or=30% GR-positive cells). CONCLUSION: Dexamethasone inhibited the growth of GR-positive cancers, possibly through the disruption of the NF-kappaB-IL-6 pathway.

DNA sequence of immunoglobulin heavy chain variable region gene in thyroid lymphoma.

Patho-epidemiological studies have shown that thyroid lymphoma (TL) develops in thyroid affected by chronic lymphocytic thyroiditis (CLTH). CLTH is categorized as an organ-specific autoimmune disease, in which activated B-lymphocytes secrete a number of autoantibodies. Because antigenic stimulation might be involved in the pathogenesis of TL, the variable region in heavy chain (V(H)) genes was characterized in 13 cases with TL and 3 with CLTH. Clonal rearrangement of the V(H) gene was found in 11 cases of TL, and cloning study with sequencing of complimentary determining region (CDR) 3 revealed the presence of a major clone in 4. Three of the 4 cases used V(H) 3 gene, with the homologous germline gene of V3-30 in two cases and VH26 in one case. A biased usage of V(H) 3 and V(H) 4 genes with the homologous germline gene of VH26 in V(H) 3 gene was reported previously in cases with CLTH. A high level of somatic mutation (1-21%, average 12%) with non-random distribution of replacement and silent mutations was accumulated in all cases. The frequency of the occurrence of minor clones ranged from 29-44% per case, indicating the presence of on-going mutation. DNA sequencing of immunoglobulin V(H) gene suggests that TL develops among activated lymphoid cells in CLTH at the germinal center stage under antigen selection.

Clonal evolution of gastric lymphoma of mucosa-associated lymphoid tissue type.

Development of multiple lesions is frequent in gastric lymphoma of mucosa-associated lymphoid tissue (MALT) type. Presence of clonal components in multiple lesions was examined on the resected samples from 18 cases by using PCR-based method for immunoglobulin heavy-chain gene rearrangement. There were two or more lesions in 10 cases, and 2 to 12 samples were obtained from each lesion. The remaining eight cases had a single large lesion, from which two to six samples were collected from separate areas from each other. A total of 86 samples were analyzed. Histologic findings in each sample were categorized as follows: proliferation of exclusively centrocyte-like cells (CCL), large cells, and combined CCL and large cells. Monoclonal or biclonal pattern (single or two bands) was observed in 42 samples, oligoclonal pattern (three or more bands) in 30, polyclonal (smear) in 11, and no products in 3. Large-cell-type lesions showed fewer bands than those with other histologic types, and 75% of cases with large-cell type had mono- or biclonal proliferation. Common clones were found among lesions in about 60% of cases. Especially in 4 cases including 2 cases with large-cell type, every lesion in the same case contained the common clones. These findings suggested that gastric MALT lymphoma started as multi- or oligoclonal proliferation of cells, in which separate lesions composed of different clones from each other. As disease advances, dominant clones appear in some lesion and disseminate to other lesions via homing properties of the proliferating B lymphocytes.

Voided urine cytology of papillary renal cell carcinoma and renal calculus: report of a case with emphasis on the importance of cytologic screening in high-risk individuals.

BACKGROUND: Preoperative diagnosis of cases of renal calculus complicated with papillary renal cell carcinoma (RCC) by image analysis is usually difficult. CASE: A 50-year-old man who had a past history of renal calculus suffered from macrohematuria and abdominal pain for one month was admitted to our hospital. Ultrasonographic examination revealed a 4-cm tumor shadow in the right kidney; it was hypovascular in arteriography. Papillary cell clusters with abundant cytoplasm were found by the cytologic examination of voided urine. Their nuclei were oval and situated eccentrically in the cytoplasm. The nuclear/cytoplasmic ratio was increased. Fine, granular chromatin was distributed evenly, and the nuclear membrane was thin and nearly smooth. Several small nucleoli were evident. All these findings were indicative of a diagnosis of papillary RCC. Histology of nephrectomy specimens confirmed the diagnosis. CONCLUSION: Voided urine cytology can be useful for screening and follow-up of patients with papillary RCC.