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KAI1 was immunohistochemically detected in the lower part of fundic glands and intestinal metaplasia in non-cancerous gastric tissues, while only 11.0% (8/73) of cancerous tissues were shown to be positive. The relationship between various clinicopathological characteristics of patients with gastric cancer and KAI1 expression in their tumors was then analyzed to reveal that the incidence of positivity was 16.6% (8/48) of patients with stage Ia-IIIa and 0% (0/25) of patients with stage IIIb-IVb (P0.05) and that the incidence of peritoneal or hepatic metastasis tended to be lower in KAI1-positive tumors than in negative ones. These results suggest that decreased KAI1 expression may be related to the progression of gastric cancer.

To define T cell co-stimulatory molecules that work in the early phase of T cell activation, we established monoclonal antibodies (mAb) that inhibit or enhance T cell activation by the histiocytic leukemia cell line U937. One of the mAb, 53H5, which recognized both T cells and U937, was identified to bind to CD82 by expression cloning. Functional analyses of CD82 revealed that 1) CD82 needs to exist on both T cells and U937 for the full activation of T cells; 2) CD82 expression is up-regulated on both T cells and U937 by stimulation such as CD3 ligation or treatment with phorbol 12-myristate 13-acetate; 3) overexpression of CD82 enhances both homotypic and heterotypic cell adhesion between T cells and U937; 4) CD82 signal co-stimulates T cells and the signal works synergistically with the CD28-mediated T cell co-stimulation signal; 5) in mixed leukocyte reactions using U937 as stimulator cells, CD82 overexpression on U937 correlates with the higher allogeneicity of U937 cells. These results indicate that CD82 co-stimulates T cells not only by sending intra-T cell signals that work synergistically with CD28 signals but also by inducing enhanced T cell-antigen-presenting cell interaction.

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16‐BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca2+‐independent aggregation of B16‐BL6 cells by KAI1 cDNA transfection compared with mock transfectants (P

Using microcell-mediated transfer of a normal chromosome 11 into the highly metastatic MDA-MB-435 human breast carcinoma cell line, we previously showed that human chromosome 11 contains a metastasis-suppressor gene for breast cancer. A known metastasis-suppressor gene, kai-1, and a related family member, tapa-1, have been mapped to chromosome 11p11.2 and 11p15.5, respectively. To determine if these genes are responsible for the metastasis suppression seen in our microcell hybrids, we examined their expression by western blot analysis. Although tapa-1 expression did not significantly correlate with metastasis suppression, kai-1 production was dramatically increased in the metastasis-suppressed chromosome 11 microcell hybrids and unchanged in the metastatic chromosome 6 controls. Transfection of full-length kai-1 cDNA into MDA-MB-435 cells resulted in clones that did not have a significantly decreased in vivo incidence of lung metastases. However, western blot analysis showed that the primary tumors and the metastatic lesions of the transfectants had decreased levels of kai-1 protein compared with the inoculated cells. Furthermore, several of the transfectant clones expressed heavily modified kai-1 protein compared with that of the microcell hybrids. Our data indicate that protein modification may affect the normal function of kai-1 in vivo and that a threshold level of kai-1 protein expression may be necessary for suppression of the metastatic phenotype.

Both biochemical and live cell imaging studies suggest the existence of lipid rafts, specialized membrane microdomains that promote interaction among signaling molecules. Although their composition is still poorly understood, these highly dynamic domains are enriched in cholesterol, sphingolipids, and particular groups of proteins. The mechanism(s) by which trafficking into or out of lipid rafts affects signaling remains unclear.

CD82 is a tetraspan transmembrane protein on NK/LAK-susceptible targets. A single highly glycosylated protein of heterogeneous molecular mass (50-90 kDa) was immunoprecipitated by anti-CD82 from Nonidet P-40 lysates of various B cell lines, Raji, Daudi, 721, and 721.134. Using the milder detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), additional proteins were coprecipitated with CD82 from surface iodinated B cell lines, including a major band at 45 kDa, identified as the HLA class I heavy chain by sequential immunoprecipitations and sequential immunoprecipitation-Western blot analysis. Cocapping experiments confirmed the molecular association of CD82 and HLA class I at the cell surface of these B cell lines. CD82 could be coprecipitated with both mature and beta 2-microglobulin (beta 2m)-free heavy chains of MHC-I from CHAPS extracts. No association between MHC-I and CD82 was found in the beta 2m-deficient Daudi cell line or after co-in vitro translation of CD82, MHC heavy chain, and beta 2m mRNA. The most likely source of free class I heavy chains at the cell surface is by dissociation of beta 2m-associated class I molecules. These results suggest that association of CD82-MHC-I takes place at the cell surface and could interfere with the capacity of the MHC-I complex to protect targets from NK-mediated cytotoxicity.

Invasion of trophoblasts into maternal uterine tissue is essential for establishing mature feto-maternal circulation. The trophoblast invasion associated with placentation is similar to tumor invasion. In this study, we investigated the role of KAI1, an anti-metastasis factor, at the maternal-fetal interface during placentation. Mouse embryos were obtained from gestational days 5.5 (E5.5) to E13.5. Immunohistochemical analysis revealed that KAI1 was expressed on decidual cells around the track made when a fertilized ovum invaded the endometrium, at days E5.5 and E7.5, and on trophoblast giant cells, along the central maternal artery of the placenta at E9.5. KAI1 in trophoblast giant cells was increased at E11.5, and then decreased at E13.5. Furthermore, KAI1 was upregulated during the forskolinmediated trophoblastic differentiation of BeWo cells. Collectively, these results indicate that KAI1 is differentially expressed in decidual cells and trophoblasts at the maternal-fetal interface, suggesting that KAI1 prevents trophoblast invasion during placentation. [BMB Reports 2013; 46(10): 507-512]

The aim of the present study was to investigate the effects of PTEN and KAI1 gene overexpression on the proliferation, metastasis and radiosensitivity of ASPC‑1 pancreatic cancer cells under hypoxic conditions. Recombinant vectors that overexpress PTEN and KAI1 genes were transfected into hypoxic ASPC‑1 cells. Protein expression levels were detected by western blot analysis. An MTT cell growth curve assay and a colony‑forming assay were used to analyze cell proliferation, and a Transwell assay was performed to evaluate the metastatic ability of the cells. Annexin V flow cytometry was used to determinate the apoptotic rate of X-ray-treated ASPC-1 cells. Western blot analysis revealed that PTEN‑ and KAI1‑transfected ASPC-1 cells significantly upregulated PTEN and KAI1 expression. The proliferation of hypoxic ASPC-1 cells was significantly suppressed by PTEN and KAI1. Furthermore, PTEN and KAI1 overexpression inhibited the metastatic ability of hypoxic ASPC‑1 cells. Following X‑ray treatment, the percentage of apoptotic cells increased significantly in the ASPC-1 cells transfected with PTEN and KAI1, which demonstrated that hypoxic ASPC-1 cells were more radiosensitive due to PTEN and KAI1 overexpression. In conclusion, double overexpression of PTEN and KAI1 inhibited the proliferation and metastatic activity, and enhanced apoptosis induced by X‑ray in ASPC‑1 cells under hypoxic conditions, which indicates that PTEN and KAI1 double-expression may have valuable application in pancreatic cancer gene therapy.

To explore the expressions of CD133 and CD82/KAI1 in bladder urothelial carcinoma, their association with the clinicopathological factors and their roles in vasculogenic mimicry (VM) in the tumor.The expressions of CD133 and CD82/KAI1 and VM were detected by immunohistochemistry and histochemistry in 90 specimens of bladder urothelial carcinoma and 20 specimens of normal bladder epithelium tissue.The positivity rates of CD133, CD82/KAI1 and VM in normal bladder epithelium tissue were 0, 90% and 0, showing significant differences from the rates of 65.6%, 31.1% and 31.1% in urothelial carcinoma, respectively (P0.01). Positive expressions of CD133, CD82/KAI1 and VM were significantly correlated with pTNM stage and tumor relapse (P0.01) but not with gender, age, or tumor numbers (P0.05). CD133 expression was positively correlated with VM (P=0.487, P0.05), and CD82/KAI1 expression was negatively correlated with VM (r=-0.452, P0.01) and CD133 (r=-0.776, P0.05).The expressions of CD133 and CD82/KAI1 proteins are involved in the occurrence of VM in bladder urothelial carcinoma to contribute to the invasion and relapse of bladder carcinoma.

MiR-195, which exhibits a proliferation-inhibiting role in different tumors, has been reported to be down-regulated in the ectopic endometrium. The aim of this study was to determine the impact of miR-195 on the biological characteristic of the endometrial stromal cells (ESCs). MiR-195 has been presumed to target the 3’-untranslated regions (3’-UTR) of Fractalkine (FKN), which also plays important roles in endometriosis. Fluorescence reporter assays showed that miR-195 effectively binds to the 3’-UTR of FKN. The normal ESCs showed a significant higher miR-195 expression than that of eutopic and ectopic ESCs associated with endometriosis, while the FKN expression showed opposite results. MiR-195 mimics inhibited proliferation and growth and induced apoptosis of eutopic ESCs, and these effects were abolished by FKN-siRNA. miR-195 could decrease the expression of survivin, matrix metalloproteinase-9 (MMP9) and up-regulate the expression of CD82, tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2 of eutopic ESCs by targeting FKN. Our study has demonstrated for the first time that miR-195 plays important roles in regulating the functions of ESCs through targeting FKN. The information may be useful for developing a new therapeutic strategy for endometriosis.

P63 is a p53 family member involved in multiple facets of biology, including embryonic development, cell proliferation, differentiation, survival, apoptosis, senescence and aging. The p63 gene encodes multiple protein isoforms either with (TAp63) or without (ΔNp63) the N-terminal transactivation domain. Amounting evidence suggests that p63 can function as a tumor suppressor, yet the precise molecular mechanisms, and particularly the specific roles of TAp63 and ΔNp63 in cancer progression, are still largely unclear. Here, we demonstrated that ΔNp63α, the predominant isoform expressed in epithelial cells and squamous cell carcinomas, inhibits cell invasion. Affymetrix gene expression profiling, combined with gain- and loss-of-function analyses and chromatin immunoprecipitation, indicated that cluster of differentiation 82 (CD82), a documented metastasis suppressor, is a direct transcriptional target of ΔNp63α. Expression of ΔNp63α inhibited outgrowth in Matrigel and cancer cell invasion, which was largely reversed by specific ablation of CD82. Conversely, ΔNp63α knockdown led to increased cell invasion, which was reversed by ectopic expression of CD82. Moreover, inhibition of glycogen synthase kinase-3β (GSK3β) by either pharmacological inhibitors or by RNA interference resulted in the downregulation of ΔNp63α and CD82 expression, concomitant with increased cell invasion, independently of β-catenin. Furthermore, decreased expression of p63 and CD82 is correlated with cancer progression. Taken together, this study reveals that ΔNp63α upregulates CD82 to inhibit cell invasion, and suggests that GSK3β can regulate cell invasion by modulating the ΔNp63α–CD82 axis.

The current study explored the expression of KAI1/CD82 and MRP1/CD9 and its significance in laryngeal squamous cell carcinoma (LSCC).The expression levels of KAI1/CD82 and MRP1/CD9 in 100 LSCC tissue specimens, as well as in 30 para-LSCC non-carcinomatous tissue specimens randomly taken from the patients, were assessed using the quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry and correlations with pathological parameters of LSCC and their influence on survival function were analyzed.KAI1/CD82 and MRP1/CD9 showed basically consistent changes in both mRNA and protein expression. Their expression in the 30 LSCC specimens was significantly lower compared with that in the corresponding non-carcinous tissues (P0.01 or 0.05), notably correlating with TNM stage, differentiation degree, clinical stage, and lymphatic metastasis (P0.01 or 0.05), but not gender, age, and LSCC growth sites (P0.05). The median survival of patients with positive KAI1/CD82 and MRP1/CD9 protein expression was longer than that of patients with negative protein expression (P0.01 or 0.05). KAI1/CD82 protein expression negatively correlated with MRP1/CD9 protein expression in LSCC (χ(2) = 31.25, P0.01).KAI1/CD82 and MRP1/CD9 may jointly participate in the development of LSCC. They may serve as the markers for judging the infiltration, metastasis, and prognosis of LSCC.

The aim of this study is to investigate the associations between KAI1/CD82 gene polymorphisms and colorectal cancer (CRC)-risk predisposition. We undertook a case–control study to analyze two KAI1/CD82 polymorphisms (exon 3 −29166 C>T and exon 9 −52840 C>A) in an Han Chinese population, by extraction of genomic DNA from the peripheral blood of 356 patients with CRC and 378 control participants, and performed KAI1/CD82 genotyping using DNA sequencing. The obtained results indicated that overall, no statistically significant association was observed in exon 9 (−52840 C>A). Nevertheless, exon 3 (−29166 C>T) genotype was at increased risk of CRC (P = 0.006; odds ratio = 1.299, CI 95 % 1.058–1.549). Furthermore, −29166 T allele CRCs were more significantly common in patients with tumor size of >4 cm than C allele CRC and in cases of poor differentiation and advanced pathological stage. These findings led us to conclude that polymorphism in exon 3 (−29166 C>T) was observed to be associated with susceptibility of CRC. However, exon 9 (−52840 C>A) polymorphism showed no correlation to CRC susceptibility. Nevertheless, further investigation with a larger sample size is needed to support our results.

To study the expression of galectin 3 (Gal-3) and CD82/KAI1 proteins in non-small cell lung cancer (NSCLC) and the correlation between their expression and clinical significance.The expression of Gal-3 and CD82/KAI1 proteins was detected by immunohistochemistry in 160 specimens of NSCLC and 20 specimens of normal lung tissue.The positive rates of Gal-3 and CD82/KAI1 proteins in the NSCLC were 63.8% and 37.5%, respectively, the positive rates of Gal-3 and CD82/KAI1 proteins in the normal lung tissue were 25.0% and 95.0%, respectively, and there was a significant difference between the two groups (P0.01). The expression of Gal-3 and CD82/KAI1 proteins was significantly correlated with the grade of tumor, lymph node metastasis, and pathological-TNM stages (all P0.05). Spearman analysis showed that there was a negative correlation between expressions of Gal-3 and CD82/KAI1 in NSCLC (r = -0.732, P0.01). Overexpression of Gal-3 and low expression of CD82/KAI1 were related to poor prognosis: the survival rate was significantly lower in the positive Gal-3 group (survival time: 23.0 ± 17.5 months) than that in the negative group (survival time: 71.6 ± 21.6 months) (P0.01). The survival rates of the CD82/KAI1-positive group (survival time: 72.5 ± 19.5 months) and CD82/KAI1-negative group (survival time: 21.6 ± 16.1 months) were significantly different (P0.01). Multivariate analysis indicated that pTNM stage and positive expression of Gal-3 and CD82/KAI1 are independent prognostic factors of NSCLC (P0.01).The expression of Gal-3 and CD82/KAI1 may be related to the initiation, development and metastasis of NSCLC. Combined detection of Gal-3 and CD82/KAI1 has an important role in predicting the progression and prognosis of NSCLC.

The metastasis suppressor CD82/KAI-1, which is a member of the tetraspanin superfamily, has been proposed to exert its activity together with glycosphingolipids. However, the mechanism of CD82 inhibition has not been fully elucidated. The present study aimed to investigate the synergistic inhibition of cell migration by the tetraspanin CD82 and gangliosides and to correlate this inhibition with activation of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (HGFR/cMet) in Hepa1-6 cell lines, whose motility and migration is stimulated by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in vitro. We found that Hepa1-6 cells transfected with the CD82 gene exhibited decreased migration in response to EGF and HGF. EGF-stimulated phosphorylation of EGFR at Tyr1173 was inhibited in these cells, which contributed to the attenuation of EGFR. Ectopic expression of CD82 in Hepa1-6 cells inhibited HGF-stimulated tyrosine phosphorylation of cMet at Tyr1313 and Tyr1365 without affecting the expression of cMet. These inhibitory effects were enhanced when CD82 was introduced with Ganglioside GM3 alone or GM2/GM3. Reduction of CD82 expression by RNA interference together with depletion of glycosphingolipids with P4 significantly enhanced cell motility and increased the expression of EGFR and its phosphorylation at Tyr1173 in response to EGF. Increased cell motility and HGF-dependent activation of cMet at Tyr1313 and Tyr1365 resulted from decreased CD82 levels and increased GM3. Furthermore, CD82 expression selectively attenuated EGFR and cMet signalling via phosphatidylinositol 3-kinase/Akt but had no affect on the activity of the MAPK signalling pathway. These results suggest that the synergistic effects of CD82 and GM3 or GM2/GM3 on EGFR expression and phosphorylation and cMet activation are responsible for CD82 inhibition of EGF- and HGF-dependent cell motility and migration of Hepa1-6 cells.

Mechanisms that regulate oligodendrocyte (OL) precursor migration and differentiation are important in normal development and in demyelinating/remyelinating conditions. We previously found that the tetraspanin CD82 is far more highly expressed in O4+OL precursors of the adult rat brain than those of the neonatal brain. CD82 has been physically linked to cMet, the hepatocyte growth factor (HGF) receptor, in tumor cells, and this interaction decreases downstream signaling. We show here that CD82 inhibits the HGF activation of cMet in neonatal and adult rat OL precursors.CD82expression is sufficient to allow precursor differentiation into mature OLs even in the presence of HGF. In contrast,CD82downregulation in adult O4+/CD82+cells inhibits their differentiation, decreases their accumulation of myelin proteins, and causes a reversion to less mature stages.CD82expression in neonatal O4+/CD82−cells also blocks Rac1 activation, suggesting a possible regulatory effect on cytoskeletal organization and mobility. Thus, CD82 is a negative regulator of HGF/cMet during OL development and overcomes HGF inhibitory regulation of OL precursor maturation.

Background Presently, CD133 is one of the hottest markers to characterize cancer stem cells and KAI1/CD82 is reported as an important marker for the metastasis and prognosis of many cancers. The purpose of our study is to explore the relationship between cancer stem cells (CSCs) marked by CD133 and KAI1/CD82 expression and the clinicopathological characteristics of patients with laryngeal squamous cell carcinoma (LSCC). Methods Immunohistochemical analysis was used to detect the expression of CD133 and KAI1/CD82 in 83 archival surgical specimens of human LSCC and 83 cases of normal laryngeal tissues. Results In LSCC, positive rates of 49.4% and 41.0% were obtained for CD133 and KAI1/CD82, respectively. The expression of CD133 in LSCC tissues was significantly higher than that in normal tissues (P < 0.001), and the expression of CD133 was positively associated with pTNM stage (P = 0.005), pathological grade (P = 0.001), and lymph node metastasis (P < 0.001). The reduced expression of KAI1/CD82 was present in LSCC tissues. The positive rate of KAI1/CD82 expression was negatively correlated with pTNM stage (P = 0.014), pathological grade (P < 0.001), and lymph node metastasis (P = 0.007). A correlation analysis showed that there was a negative relationship between the expression of CD133 and KAI1/CD82 protein in LSCC tissues (P < 0.001). By Kaplan-Meier analysis, the expression of CD133 was negatively correlated with overall survival (OS) (log-rank = 40.949, P < 0.001) and disease-free survival (DFS) (log-rank = 39.307, P < 0.001) time of LSCC. The expression of KAI1/CD82 was positively correlated with OS (log-rank = 40.279, P < 0.001) and DFS (log-rank = 39.271, P < 0.001) time of LSCC. Cox regression analysis: the expression of CD133 and KAI1/CD82, and pTNM stages were independent prognostic factors of LSCC (P < 0.05). Conclusions Thus the detection of CD133 and KAI1/CD82 proteins may be used as a potential indicator of LSCC prognosis.

Objective: This study aimed to investigate the expressions and significance of KAI1/CD82 and CyclinD1 in laryngeal squamous cell carcinoma (LSCC). Methods: Real-time quantitative PCR (Q-PCR) and Western blot assay were employed to detect the expressions of KAI1/CD82 and CyclinD1 in the laryngeal tissues of 86 LSCC patients, 32 patients with laryngeal polyp and 38 patients with laryngeal leukoplakia, and the influence of both proteins on the clinicopathological features and survival of LSCC patients. Results: The changes in mRNA and protein expressions of KAI1/CD82 and CyclinD1 were consistent in three groups, and the expressions of KAI1/CD82 and CyclinD1 were significantly different among three groups (P0.05). In LSCC patients positive for KAI1/CD82 protein expression, the median survival time was 76 months, which was significantly longer than that in LSCC patients negative for KAI1/CD82 protein expression (48 months; X2=16.293, P=0.000). The CyclinD1 expression in patients with TNM stage III-IV LSCC, poorly differentiated LSCC, or clinical stage III-IV LSCC was dramatically higher than that in patients with TNM stage I-II LSCC, well differentiated LSCC, or clinical stage I-II LSCC (P0.05). In LSCC patients positive for CyclinD1 protein expression, the median survival time was 40 months, which was markedly shorter than that in LSCC patients negative for CyclinD1 protein expression (X2=9.517, P=0.02). In LSCC patients, there was a negative correlation between KAI1/CD82 expression and CyclinD1 expression (X2=7.86, P

The KAI1/CD82 tetraspanin is a widely expressed cell surface molecule thought to organize diverse cellular signaling processes. KAI1/CD82 suppresses metastasis but not tumorigenicity, establishing it as one of a class of metastasis suppressor genes. In order to further assess its functions, we have characterized the phenotypic properties of Kai1/Cd82 deleted mice, including viability, fertility, lymphocyte composition, blood chemistry and tissue histopathology, and of their wild-type and heterozygote littermates. Interestingly, Kai1/Cd82(-/-) showed no obvious genotype associated defects in any of these processes and displayed no genotype associated histopathologic abnormalities after 12 or 18 months of life. Expression profiles of non-immortal, wild-type and Kai1/Cd82(-/-) mouse embryo fibroblast (MEFs) indicated distinct sex-specific and genotype-specific profiles. These data identify 191 and 1,271 differentially expressed transcripts (by twofold at P < 0.01) based on Kai1/CD82 genotype status in female and male MEFs, respectively. Differentially expressed genes in male MEFs were surprisingly enriched for cell division related processes, suggesting that Kai1/Cd82 may functionally affect these processes. This suggests that Kai/Cd82 has an unappreciated role in the early establishment of proliferation and division when challenged with a new environment that might play a role in adaptability to new metastatic sites.

We have previously shown that the presence of trophoblast cells enhances invasiveness of decidualizing human endometrial stromal cells. The metastasis suppressor CD82, which has antimigratory function in tumor cells, is up-regulated in decidualizing endometrial stromal cells. CEACAM1 is expressed in trophoblast cells at the invasion front in early placenta and is considered proinvasive. Here, we investigate the role of CD82 and CEACAM1 in cocultures of the endometrial stromal cell line T-HESC and AC-1M88 trophoblast cells. In transwell migration assays, chemotaxis of AC-1M88 cells was stimulated by coplated T-HESC in the lower compartment or by the combination of heparin-binding EGF-like growth factor (HB-EGF), interleukin-1 beta (IL-1beta), and leukemia inhibitory factor (LIF), local factors present at the time of implantation. In an implantation model of AC-1M88 trophoblast spheroids on a monolayer of T-HESC, spheroid expansion was enhanced in the presence of HB-EGF/IL-1beta/LIF. Silencing of CEACAM1 in AC-1M88 blunted this response. Chemotactic migration of T-HESC was stimulated by trophoblast secretions or HB-EGF/IL-1beta/LIF. These responses were suppressed by CD82 depletion in T-HESC. Proteome profiling revealed the presence of platelet-derived growth factor (PDGF)-AA in trophoblast supernatant. Chemotaxis of T-HESC toward PDGF-AA was significantly inhibited by CD82 silencing. Neutralization of PDGF-AA in AC-1M88 conditioned media reduced the chemotactic effect on T-HESC. In summary, we demonstrate a mutual stimulation of chemotactic migration between trophoblast and endometrial stromal cells and promigratory roles for the cell surface molecules CEACAM1 and CD82, which may serve to support tissue remodeling at the implantation site.

We have previously demonstrated that expression of COX-2 is upregulated by hepatocyte growth factor in thyroid papillary carcinoma (TPC) cells and is associated with increased invasiveness of tumor cells. COX-2 upregulation was associated with downregulation of KAI-1/CD82, a metastasis suppressor molecule that has been associated with the metastatic potential of several solid tumors. In the present study, we have investigated the possibility that downregulation of KAI-1/CD82 may contribute to the invasiveness of papillary carcinoma cells.Expression of KAI-1/CD82 and its relation to COX-2 levels were investigated in 6 primary cultures of TPC, in 2 tumor cell lines (TPC-1 and K1), and in 55 tumor samples of TPC. The biological role of KAI-1/CD82 in regulating tumor invasiveness was investigated in TPC cell lines and primary cultures transfected with a pCDNA3.1/Hygro.KAI-1; transfected cells were tested in functional studies of cell migration and invasiveness. Finally, the role of KAI-1/CD82 in influencing TPC metastasis was investigated in vivo using nu/nu mice injected with K1-transfected cells.We provide evidence that COX-2 and KAI-1/CD82 are inversely regulated in TPC primary cultures and in TPC-1 tumor cells. In fact, inhibition of COX-2 with NS398 is associated with a 2-9-fold upregulation of KAI-1/CD82 RNA. Moreover, a possible relation between COX-2 and KAI-1/CD82 was confirmed by the presence of a statistically significant inverse correlation in the expression of the two genes in 55 tumor samples of TPC (r = -0.513; p = 0.001). In 36 of 55 cases, tumor areas contained lower levels of KAI-1/CD82 RNA as compared with the corresponding normal tissue. Low expression of KAI-1/CD82 RNA in the tumor area was associated with extrathyroid extension of the disease in 16 of 19 cases (p0.04) and with lymph node metastasis in 11 of 14 cases (not significant). KAI-1/CD82 re-expression in tumor cells was associated with a significant decrease in their migratory (50-76% reduction) and invasive (46-65% reduction) capacity, even after hepatocyte growth factor stimulation. Finally, nu/nu mice injected with KAI-1/CD82-transfected K1 TPC cells developed fewer and smaller metastasis as compared with mice injected with vector-transfected K1 cells (p=0.016).Our findings raise the possibility that downregulation of KAI-1/CD82 in TPC cells is one of the molecular mechanisms regulating their invasive and metastatic potential.

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galβ1,4GlcNAc-R (Galα1,3-Gal)–dependent and –independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal–independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal–independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal–independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major “xenogenicity sensor” and open new avenues of intervention to making xenotransplantation a clinical reality.

CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR antigens. Moreover, the VLA integrins and HLA-DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the co-precipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS-7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single-transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA-DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre-existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.

To determine how tetraspanin KAI1/CD82, a tumor metastasis suppressor, inhibits cell migration, we assessed which cellular events critical for motility are altered by KAI1/CD82 and how KAI1/CD82 regulates these events. We found that KAI1/CD82-expressing cells typically exhibited elongated cellular tails and diminished lamellipodia. Live imaging demonstrated that the polarized protrusion and retraction of the plasma membrane became deficient upon KAI1/CD82 expression. The deficiency in developing these motility-related cellular events was caused by poor formations of actin cortical network and stress fiber and by aberrant dynamics in actin organization. Rac1 activity was reduced by KAI1/CD82, consistent with the diminution of lamellipodia and actin cortical network; while the growth factor-stimulated RhoA activity was blocked by KAI1/CD82, consistent with the loss of stress fiber and attenuation in cellular retraction. Upon KAI1/CD82 expression, Rac effector cofilin was not enriched at the cell periphery to facilitate lamellipodia formation while Rho kinase exhibited a significantly lower activity leading to less retraction. Phosphatidylinositol 4, 5-biphosphate, which initiates actin polymerization from the plasma membrane, became less detectable at the cell periphery in KAI1/CD82-expressing cells. Moreover, KAI1/CD82-induced phenotypes likely resulted from the suppression of multiple signaling pathways such as integrin and growth factor signaling. In summary, at the cellular level KAI1/CD82 inhibited polarized protrusion and retraction events by disrupting actin reorganization; at the molecular level, KAI1/CD82 deregulated Rac1, RhoA, and their effectors cofilin and Rho kinase by perturbing the plasma membrane lipids.

SPECIFIC AIMSThe down-regulation of apoptosis is a hallmark of tumorigenesis. We wanted to investigate how the metastasis suppressor C33/CD82/KAI1 is able to induce cell death.PRINCIPAL FINDINGS1. The metastasis suppressor gene C33/CD82/KAI1 is an apoptosis inducerUsing a screen for apoptosis-inducing genes, we detected the metastasis suppressor gene C33/KAI1/CD82. CD82 is a gene that is down-regulated in many metastatic tumor cells and whose expression can attenuate the process of metastases formation in a variety of tumors. We observed cell death induction by CD82 in many different cell types. Moreover, the apoptosis inducer cycloheximide was >threefold stronger in cells with reconstituted CD82 expression compared with CD82 negative cells. This indicates that CD82 mediates a proapoptotic signal caused by specific stimuli. As CD82 is a transmembrane protein, it was speculated that it mediates its anti-metastatic effect by interacting with components of the basal membrane. When we deleted the long, glycos...

This study investigates the expression of CD82/KAI1 and epithelial-cadherin (E-cad) in non-small cell lung cancer (NSCLC).Tissues of resected primary NSCLC and normal lung tissue were investigated. Protein expression was detected with immunohistochemical staining. mRNA expression levels of CD82/KAI1 and E-cad were determined by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) after mRNA extraction.The expression of CD82/KAI1 and E-cad was significantly lower in NSCLC compared to normal lung tissue (P0.01). CD82/KAI1 and E-cad mRNA and protein expression were found to be in close relationship with the grade of differentiation, lymph node metastasis and pathologic tumor-node-metastasis (pTNM) stages, and survival in NSCLC (P0.01), but no relationship was observed with gender, diameter, type, histological type and age (P0.05). Expression of mRNA CD82/KAI1 and E-cad were consistent with their proteins (P0.01), and there was a significant relationship between expression of CD82/KAI1 and E-cad. The survival rate of the CD82/KAI1-positive and CD82/KAI1-negative groups was significantly different (P0.01). In addition, the survival rate was significantly different between the E-cad-positive and E-cad-negative groups. pTNM stages and positive expression of CD82/KAI1 and E-cad were independent prognostic factors of NSCLC (P0.05).Lower expression of CD82/KAI1 and E-cad was found in NSCLC compared to normal lung tissue. Decreased expression of CD82/KAI1 and E-cad was closely related to cellular differentiation, pTNM stages, invasion and metastasis.

We recently reported that adhesion molecule CD82 is aberrantly expressed in CD34(+) /CD38(-) leukemia stem cells (LSCs). Here, we report the results of a functional analysis of CD82 in CD34(+) /CD38(-) acute myelogenous leukemia (AML) cells. Short hairpin (sh)RNA-mediated downregulation of CD82 resulted in a decrease in the level of IL-10. In contrast, forced expression of CD82 in CD34(+)/CD38(+) AML cells by transduction with CD82-expressing lentiviral particles resulted in an increase in the levels of IL-10. Notably, exposure of CD34(+)/CD38(-) AML cells to IL-10 stimulated clonogenic growth of these cells. Moreover, downregulation of CD82 by a shRNA dephosphorylated STAT5 in CD34(+)/CD38(-) AML cells. On the other hand, forced expression of CD82 resulted in increase in the levels of p-STAT5 in CD34(+)/CD38(+) AML cells. Chromatin immunoprecipitation (ChIP) assay results indicated that STAT5A binds to the promoter region of the IL-10 gene, while reporter gene assay results indicated stimulation of IL-10 expression at the transcriptional level. These results suggest that CD82 positively regulates the STAT5/IL-10 signaling pathway. Moreover, shRNA-mediated downregulation of CD82 expression in CD34(+)/CD38(-) AML cells dephosphorylated STAT5 in immunodeficient mice. Taken together, our data suggest that the CD82/STAT5/IL-10 signaling pathway is involved in the survival of CD34(+)/CD38(-) AML cells and may thus be a promising therapeutic target for eradication of AML LSCs.

In recent years, many studies have revealed the prognosis in patients with non-small cell lung cancer (NSCLC). In general, some clinic-pathological parameters have been related with prognosis. The aim of this study is to detect the relationship among lymphatic vessel density (LVD), microvessel density (MVD), expression of carcinoembryonic antigen (CEA) mRNA, metastasis suppressor genes (KAI1 and Kiss-1), and the prognosis of NSCLC patients.Blood samples were collected from 57 cases of NSCLC. The transcription of CEA mRNA was detected via nested reverse transcriptase-polymerase chain reaction and micro-fluid chip. Immunohistochemistry was performed to detect the expression of LVD, MVD, KAI1 and Kiss-1 in the patients. All follow-up data were collected and analyzed.The overall five-year survival rate was 18%, and the median survival was 34 months. TNM stage, lymph node metastasis, and expression of MVD, LVD, CEA mRNA and Kiss-1 were factors to survival, as determined via single survival analysis. Multivariate analysis demonstrated that TNM stage, lymph node metastasis, and expression of CEA mRNA were independent prognostic factors for NSCLC patients.The expression of MVD, LVD, Kiss-1 and CEA mRNA is related to the prognosis of NSCLC.

Our previous work has demonstrated that cyclosporin A (CsA) up-regulates but CD82 down-regulates the invasiveness of human trophoblasts. In the present study, we further investigated whether CsA can modulate the trophoblasts invasion through regulating the expression of CD82 in decidual stromal cells (DSCs). A co-culture model was established to investigate the effect of CsA on trophoblasts invasiveness. In-cell Western was performed to evaluate the expression of CD82, p53, β-catenin and the phosphorylation level of NF-κB p50 in DSCs. The secretion of CXCL12 of trophoblasts and DSCs was determined by enzyme-linked immunosorbent assay (ELISA). We found that CsA could not directly change the expression of CD82 in DSCs, but the CsA-treated trophoblasts significantly enhanced CD82 expression, NF-κB p50 phosphorylation and p53 expression, and decreased β-catenin expression in DSCs, and these effects could be abolished by anti-CXCL12 or CXCR4 neutralizing antibody. In addition, the invasiveness of trophoblast cells was markedly decreased after blocking CXCR4 of trophoblasts. Interestingly, when DSCs were pretreated with anti-CXCR4 neutralizing antibody, the invasiveness of trophoblast cells was enhanced in the coculture unit, and blocking CXCR4 on DSCs could reverse the decrease of trophoblasts invasiveness induced by CD82. Moreover, CsA further amplified these effects mediated by CXCL12 and CD82. Our results suggest that CsA not only promotes the trophoblasts invasiveness through stimulating the secretion of CXCL12, but also limits the invasiveness of trophoblasts by indirectly up-regulating the expression CD82. Therefore, CsA may contribute to the appropriate invasiveness of trophoblasts via strengthening the crosstalk between trophoblasts and DSCs.

To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated CD34(+) /CD38(-) cells with that of CD34(+) /CD38(+) counterparts from individuals with acute myelogenous leukemia (n = 2, AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in CD34(+) /CD38(-) AML cells compared with their CD34(+) /CD38(+) counterparts. Proteins overexpressed in CD34(+) /CD38(-) AML cells included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, antiapoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in CD34(+) /CD38(-) AML cells was noted in additional clinical samples (n = 12) by flow cytometry. Importantly, down-regulation of CD82 in CD34(+) /CD38(-) AML cells by a short hairpin RNA (shRNA) inhibited adhesion to fibronectin via up-regulation of matrix metalloproteinases 9 (MMP9) and colony forming ability of these cells as assessed by transwell assay, real-time RT-PCR, and colony forming assay, respectively. Moreover, we found that down-regulation of CD82 in CD34(+) /CD38(-) AML cells by an shRNA significantly impaired engraftment of these cells in severely immunocompromised mice. Taken together, aberrant expression of CD82 might play a role in adhesion of LSCs to bone marrow microenvironment and survival of LSCs. CD82 could be an attractive molecular target to eradicate LSCs.

Intensive protein synthesis is a unique and differential trait of multiple myeloma (MM) cells. Previously we showed that tetraspanin (CD81, CD82) overexpression in MM cell lines attenuated Akt/mTOR cascades, activated UPR, and caused autophagic death, suggesting breach of protein homeostasis. Here, we explored the role of protein synthesis in the tetraspanin-induced MM cell death. Contrary to attenuation of the major metabolic regulator, mTOR we determined elevated steady-state levels of protein in CD81N1/CD82N1 transfected MM lines (RPMI-8226, CAG). Elevated levels of immunoglobulins supported increased protein production in RPMI-8226. Changes in cell morphology consistent with elevated protein synthesis were also determined (cell, nuclei, and nucleoli sizes and ratios). Increased levels of phospho-rpS6 and decreased levels of phospho-AMPK were consistent with increased translation but independent of mTOR. Involvement of p38 and its role in tetraspanin induced translation and cell death were demonstrated. Microarray analyses of tetraspanin transfected MM cell lines revealed activation of protein synthesis signaling cascades and signals implicated in ribosome biogenesis (snoRNAs). Finally, we showed tetraspanins elevated protein synthesis was instrumental to MM cells' death. This work explores and demonstrates that excessive protein translation can be detrimental to MM cell lines and therefore may present a therapeutic target. Proteostasis is particularly important in MM because it integrates the high levels of protein production unique to myeloma cells with critically important microenvironmental cues. We suggest that increasing translation may be the path of least resistance in MM and thus may afford a novel platform for strategically designed therapy. J. Cell. Biochem. 113: 2500–2510, 2012. © 2012 Wiley Periodicals, Inc.

Background KAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression. Methods We used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 μg/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth in vivo and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level. Results We demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an in vivo xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α. Conclusions These novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.

Head and neck cancer is included among the top five most commonly prevailing cancers worldwide. Abnormalities of either genetic or epigenetic factors are found responsible for the development and progression of head and neck cancer. Metastasis is the leading cause of death in patients with head and neck cancer. Down regulation of metastasis suppressor genes (MSGs) expression have been frequently observed in advanced tumours.The present study was designed to screen two of the most frequently down-regulated MSGs (KISS1 and KAI1) for mutations in 120 diagnosed head and neck cancer affected Pakistani patients. The questionnaire was filled for basic information about age, gender, smoking habits and area of cancer affected and other relevant details. Primers for both genes were designed using "Primer 3" software in such a way that both intron exon boundaries were included in this region. DNA isolation and estimation was done by using organic method and agarose gel electrophoresis. Single Strand conformational polymorphism technique was used after amplification of the respective genes. Mobility patterns were analyzed using BioDoc Analyzer.Data of patients were analyzed on the basis of age, sex and type of cancer as variables. The mean age of patients and controls was 44 years. There were 53% females and 47% males in this group of study, 63% nonsmokers and 37% smokers and larynx cancer was found to be most frequent type of cancer with a percentage of 64. Lack of germ line mutation was observed in the entire coding region in both coding regions as well as splice sites of the respective genes.Germ line mutations in KISS1 and KAI1 are thus considered to be a less frequent event in head and neck cancer patients. However, two polymorphisms in intronic region of exon 3 and exon 9 of KAI1 gene were observed in 1% of patients. In non coding region downstream of exon 3 (KAI1), there was a C 29166 T substitution and in intronic region upstream exon 9 of KAI1 gene, a C 52840 A substitution was observed. Both patients were females with ages 47 and 50 years respectively. A detailed analysis of regulatory mechanism is required to explore the genetic basis of down regulation of these MSGs for a better understanding of head and neck cancer progression.

Metastasis suppressor genes are involved in the inhibition of a cancer cell's ability to metastasize. Down expression of such genes may contribute to pathogenesis of breast cancer. The aim of current study was firstly to evaluate expression of two examples, KAI1 and KISS1, and then to determine relationships with stages of breast cancer in a Pakistani population.Fresh biopsy tissues were collected from different hospitals and oncology research institutes. The semi quantitative reverse transcriptase polymerase chain reaction was used to investigate KAI1 and KISS1 expression in 25 breast tumor tissues and 25 normal tissues. Statistical analysis was performed to explore its association with breast cancer risk.The present study revealed that KAI1 and KISS1 mRNA expression was markedly reduced in tissues of breast cancer compared to adjacent normal tissue. In present study a splice variant of KAI1 during a screen for its expression analysis was also observed. This splice variant has not been reported previously.Metastasis suppressor genes demonstrate reduced expression in breast cancers in Pakistan.

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γnull (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.

Background Well-controlled trophoblast invasion at maternal-fetal interface is a critical event for the normal development of placenta. CD82 is a member of transmembrane 4 superfamily, which showed important role in inhibiting tumor cell invasion and migration. We surmised that CD82 are participates in trophoblast differentiation during placenta development. Methodology/Principal Findings CD82 was found to be strongly expressed in human first trimester placental villous and extravillous trophoblast cells as well as in trophoblast cell lines. To investigate whether CD82 plays a role in trophoblast invasion and migration, we further utilized human villous explants culture model on matrigel and invasion/migration assay of trophoblast cell line HTR8/SVneo. CD82 siRNA significantly promoted outgrowth of villous explants in vitro (P

C33 Ag (CD82) is a member of the transmembrane 4 superfamily (TM4SF) and an activation Ag of T-cells. Recent studies have shown that CD82 associates with CD4 or CD8 and delivers costimulatory signals for the TCR/CD3 pathway. We have isolated cDNA and genomic clones of mouse CD82. Mouse CD82 has 266 amino acid residues with 76% identity to human CD82. The mouse CD82 gene consists of nine exons and spans more than 20 kb of genomic DNA. The genomic organization of CD82 is quite similar to that of three other TM4SF members whose genomic structures were described, i.e., Tapa-1 (CD81), CD53, and CD63. By mapping the 5′ end of CD82 transcripts, we found a single major transcription initiation site 144 bp upstream of the ATG initiation codon. We also determined the sequence of the 5′ flanking region of CD82 gene for about 2 kb. The 5′ flanking sequence has a housekeeping promoter with potential binding motifs for various transcriptional factors. Northern blot analysis showed quite variable expression of the CD82 gene among different organs. The highest expression was seen in the spleen and the kidney. The expression was low in skeletal muscle and hardly detectable in the heart. Northern blot analysis was also carried out for CD81, CD53, and CD63. The expression of the CD81 gene was ubiquitous and similar among different organs, while that of CD53 was seen only in the spleen. The expression of the CD63 gene was ubiquitous, with the highest expression in the kidney. These results together with the comparison of the structures of 5′ flanking sequences of these genes indicate distinct regulations of gene expression for these four members of TM4SF.

The mononuclear cell surface protein IA4 was originally identified in our lab using a mAb selected because of its strong reactivity with three lymphoblastoid variant cell lines which are HLA class I deficient, are LAK susceptible, and form a high number of conjugates with LAK effectors. We previously cloned the cDNA of the IA4 protein, coding for a 267-amino-acid type III integral membrane protein, with four transmembrane domains and three possible N-glycosylation sites. The IA4 protein belongs to the tetra span transmembrane (TST) new family of surface molecules, which also includes CD9, CD37, CD53, CD63, and TAPA-1. IA4 antigen was recently recognized as belonging to a new cluster of differentiation CD82 (International CD Workshop, Boston 1993). The IA4 antigen expression pattern at the surface of immune cells from normal donors was studied. On T lymphocytes, IA4 was barely detectable on resting cells and increased 3.5- to 7-fold following PHA or PHA+PMA stimulation. This IA4 increased expression is correlated with the morphologic change in blast cells and with the expression of activation markers such as CD2 and MHC class II antigens, therefore suggesting that IA4 is an activation marker on T lymphocytes. The expression of IA4 was low on circulating resting monocytes collected by elutriation. However, these monocytes, cultured in medium alone or with GM-CSF, acquired the morphology of macrophage and simultaneously overexpressed MHC Class II, CD14, and IA4 antigens, suggesting that IA4 is a differentiation marker for macrophages, whatever the culture conditions, either adherent (plastic culture dishes) or nonadherent (Teflon culture bags). IA4 stable transfectants of the murine mastocytoma cell line P815 were obtained and used to generate a new mAb. Competitive epitope binding studies have shown that IA4 antigen presents a dominant epitope recognized by most of the mAb prepared either in our lab or elsewhere. This dominant epitope is not shared by any of the other antigens of the TST family. Using this new mAb we were able to biochemically characterize the IA4 antigen as a 28-kDa protein, highly N-glycosylated with different patterns on various cells.

Vasculogenic mimicry (VM), found in many high invasive ability tumors, is associated with tumor invasion and metastasis. Many genes exhibit abnormal levels of expression in these tumors. This study aims to find good markers for predicting the invasion and metastasis of non-small cell lung cancer (NSCLC).VM and expression of CD82/KAI1 and HIF-1α were examined via immunohistochemistry and histochemistry of 160 NSCLC and 20 normal lung tissue specimens.In NSCLC, positive rates of 37.5%, 48.8%, and 36.9% were obtained for CD82/KAI1, HIF-1α, and VM, respectively. In normal lung tissue, positive rates of 95.0%, 0, and 0 were obtained for CD82/KAI1, HIF-1α, and VM, respectively. A significant difference was found between the NSCLC and normal lung groups (P0.01). VM and the expression levels of CD82/KAI1 and HIF-1α were significantly related to tumor differentiation, lymph node metastasis, clinical staging, and postoperative survival time (P0.01 for all). A negative correlation was found between the expression levels of CD82/KAI1 and HIF-1α; a similar relationship was observed between CD82/KAI1 and VM. A positive relationship between the expression of HIF-1α and VM was revealed; there was a significant relationship between microvessel density (MVD) and the expression of CD82/KAI1 or HIF-1α or VM. VM and overexpression of HIF-1α were related to poor prognosis: the survival rates were significantly lower in positive patients than in negative patients (both P0.01). The survival rates of the CD82/KAI1-positive and CD82/KAI1-negative groups were significantly different (P0.01). The five-year survival rate was significantly different between the group with MVD≥22 and the group with MVD22 (P0.01). pTNM stage, positive expressions of CD82/KAI1 and HIF-1α, and VM were independent prognostic factors of NSCLC (P0.01).VM and the expressions of CD82/KAI1 and HIF-1α in NSCLC are related to differentiation, lymph node metastasis, clinical staging, and prognosis. The combined detection of CD82/KAI1, HIF-1α, and VM has an important role in predicting the progression and prognosis of NSCLC.

To study the correlation of the expressions of CD82 and hTERT and HPV infection with the clinical pathological features of penile cancer and identify their prognostic significance in the lymphatic metastasis of the disease.A total of 44 patients underwent partial or radical penectomy and lymph node dissection. The expressions of CD82 and hTERT were determined by immunohistochemistry, and HPV infection was detected by PCR.The positive rates of CD82, hTERT, and HPV DNA in penile carcinoma were 47.7%, 38.6% and 25.9%, respectively. The amplified HPV DNA was HPV-16. The pathological stage and hTERT expression were positively correlated with inguinal lymph node metastasis of penile cancer (P = 0.032, P = 0.041), and so was the pathological stage with the expression of CD82 (P = 0.045), but neither the pathological stage, nor the expression of CD82 or the positive rate of HPV DNA showed any correlation with lymph node metastasis (P = 0.627, P = 0.094, P = 0.633).The pathological grade and hTERT expression are independent prognostic factors for lymph node metastasis in penile carcinoma. These features help the prognosis and identification of the patient at the risk of nodal metastasis.

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are 5-fluorouracil (5-FU) metabolizing enzymes and are involved in the sensitivity of carcinoma patients to 5-FU. Although 5-FU is often used for the treatment of oral carcinoma, there has not been any investigation into the expression of these enzymes in metastatic lymph nodes or of their roles in the effectiveness of 5-FU in treating lymph node-metastatic cancer. Oral squamous cell carcinoma (OSCC) often metastasizes to the lymph nodes, and these enzymes may be significant in the survival of patients with this disease. This study investigated the expression of TS and DPD in cervical lymph node metastases and its relationship with primary OSCC, as well as the interaction between these enzymes and Kangai 1(KAI1/CD82) which is a metastasis suppressor protein. Surgical specimens from 20 cases of OSCC with lymph node metastasis, 20 cases of OSCC without lymph node metastasis, and 10 cases of normal mucosa were examined by immunohistochemistry. The relationship between TS and DPD expression and clinicopathological data was analyzed. TS and DPD proteins were overexpressed in primary OSCC compared to that in normal mucosa. TS expression of the primary oral cancer cells in the group with lymph node metastasis was higher than that of those without. DPD expression did not significantly correlate with the occurrence of lymph node metastasis, nor was it different between primary oral cancer cells and cervical metastases. CD82 expression was significantly reduced in lymph node metastases. These findings indicate that TS and CD82 may be of great value in assessing lymph node metastasis of OSCC, and could be taken as new targets for therapy of metastatic OSCC.

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn(157) for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.

Tetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer cells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic implantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of endometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its receptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein levels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of the primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by downregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinβ1 signal pathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17β-estradiol can promote the invasion of ESCs via suppressing CD82 expression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2–CCR2 recruits more macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the ectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced by TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2 secretion and CCR2 expression and the invasion of ESCs via MAPK and integrinβ1 signal pathway.

KAI1/CD82, a tetraspanin membrane protein functions as a metastasis suppressor in many types of human cancers and has been shown to regulate cell adhesion properties. In the present study, we investigated the underlying mechanism of KAI1/CD82-mediated changes in cell adhesion to the extracellular matrix using human prostate cancer cells. We found that high KAI1/CD82 expression attenuated short-term cell adhesion to uncoated- or fibronectin-coated plates. Moreover, high KAI1/CD82 expression generated an extracellular environment unfavorable for cell adhesion as compared to low KAI1/CD82 expression, suggesting KAI1/CD82-dependent regulation of extracellular matrix (ECM) molecule(s) expression and/or secretion. Among ECM components examined, fibronectin exhibited decreased expression and secretion in high KAI1/CD82-expressing cells. Furthermore, high KAI1/CD82 expression interfered with the activation of β (1) integrin at the cell surface while total β (1) integrin levels remained unchanged, concomitant with reduced formation of focal adhesion complex and decreased bundling of actin filaments. Finally, high KAI1/CD82 expression significantly retarded cell motility in a scratch wound assay. Taken together, our results strongly suggest that KAI1/CD82 attenuates the activation of β (1) integrin, and thereby down-regulates outside-in signaling of β (1) integrin, leading to the reduction of focal adhesion formation and fibronectin expression/secretion, which subsequently interferes with cell adhesion properties and motility.

Background The aim of this study was to clarify the clinical significance of TM4SF members CD9, CD63 and CD82 in human gastric carcinoma. Methods By employing RT-PCR and immunohistochemistry, we studied the expression of CD9, CD63 and CD82 in 49 paired tissue specimens of normal gastric mucosa and carcinoma. All tissues were obtained from patients who underwent curative surgery. Results All normal gastric epithelium and gastric ulcer tissues strongly expressed transcripts and proteins of CD9, CD63 and CD82 as compared with corresponding controls. We found a significant correlation between CD63 mRNA level and different pM statuses (P = 0.036). Carcinomas in M0 stage revealed a stronger expression of CD63 than carcinomas in M1 stage. Expression of CD9 protein was found significantly stronger in pN0, pM0 than in advanced pN stages (P = 0.03), pM1 (P = 0.013), respectively. We found the relationship between CD63 expression, gender (p = 0.09) and nodal status (p = 0.028), respectively. Additionally, advanced and metastasized tumor tissues revealed significantly down-regulated CD82 protein expression (p = 0.033 and p = 0, respectively), which correlated with the tumor pTNM stage (p = 0.001). Conclusion The reduction of CD9, CD63 and CD82 expression are indicators for the metastatic potential of gastric carcinoma cells. Unlike their expression in other tumor types, the constitutive expression of CD63 may indicate that this factor does play a direct role in human gastric carcinogenesis.

NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFκB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade.

Tetraspanin CD82 has been identified as a potential contributor to controlling trophoblast invasiveness in human first-trimester pregnancy. However, it is unclear how the regulation of CD82 expression at maternal-fetal interface. The present study is to investigate the effect of the trophoblast-derived CXCL12 on CD82 expression in decidual stromal cells (DSCs) that in turn controls trophoblast cell invasiveness. In-cell Western was used to evaluate the expression of CD82 in DSCs. A co-culture model was established to investigate the reciprocal interaction between trophoblasts and DSCs via CXCL12/CXCR4 and CD82 expression. We found that both anti-CXCL12 and anti-CXCR4 neutralizing antibody can eliminate increase of CD82 expression in DSCs induced by the trophoblasts supernatant. Moreover, the invasiveness of trophoblasts pre-treated with anti-CXCR4 neutralizing antibody was significantly decreased. Interestingly, when DSCs were pre-treated with anti-CXCR4 neutralizing antibody, the trophoblasts invasiveness in the co-culture was enhanced, and thus anti-CXCR4 neutralizing antibody can reverse the decrease of trophoblasts invasiveness induced by CD82. The trophoblast cell-derived CXCL12 does not only increase the invasiveness in an autocrine manner, but also control the over-invasion of trophoblasts through promoting CD82 expression in DSCs in a paracrine manner, which maintains a physiological balance of human trophoblasts invasiveness via the cross-talk between trophoblasts and DSCs.

Distinction of renal oncocytoma (RO) from chromophobe renal cell carcinoma (ChRCC) is important because their clinical behavior is different. As part of a search for the best available immunohistochemical markers to distinguish ChRCC from RO, we investigated the immunohistochemical profiles of these tumors. We selected 30 renal tumors consisting of ChRCC, typical variant (n = 14), ChRCC, eosinophilic variant (n = 6), and RO (n = 10). Their expression of cytokeratin (CK) 7, KAI1, epithelial-specific antigen (ESA), epithelial-related antigen (ERA), Claudin- 7, and Claudin-8 was studied using an autostainer. Immunoreactivity was assessed based on a combined score of the extent and intensity of staining. Compared to RO, a significantly higher percentage of the total ChRCCs stained positive for CK7 (85% vs. 10%, respectively), KAI1 (90% vs. 10%), ESA (95% vs. 10%), ERA (95% vs. 10%), and Claudin-7 (95% vs. 20%) (P < 0.001). Additionally, there was a significant difference between the percentage of ChRCC eosinophilic variant (ChRCC-E) and RO that stained positive for KAI1 (100% vs. 10%, respectively), ESA (83% vs. 10%), and ERA (83% vs. 10%) (P < 0.001). We recommend immunohistochemical analysis of KAI1, ESA, and ERA to distinguish ChRCC-E from RO.

The recognition that cellular regulatory circuitry is composed of antagonistic elements has made possible a new approach to the selection of regulatory genes, called the gene-gene interference method. The method was used to identify and isolate genes possibly related in an antagonistic way to protein kinase A. Two such genes were recovered: ART1 encodes a potential regulator of cytokinesis and KAI1 appears to be involved in the Start control. The principles of the gene-gene interference method are discussed, as well as its possible general use for 'walking' within the cellular regulatory networks of eukaryotes.