Search

Tetraspanins are transmembrane adaptor proteins involved in the regulation of various fundamental cellular processes. For a number of malignant diseases, the level of expression of members of the tetraspanin family was found to correlate with tumor cell invasiveness, ability to form metastases, and poor clinical outcome. We describe the exact quantification of mRNAs coding for the tetraspanins CD9, CD63, CD82 and CD151 expressed by mammary carcinoma-derived cell lines that were classified as invasive or non-invasive according to their ability to penetrate collagen-fibroblast gels in vitro. The mean of beta2-microglobulin-normalized expression of CD9 was about 10-fold higher than the mean calculated for CD63 and about 20-fold higher than expression of CD82 and CD151. Direct comparison of tetraspanin expression of invasive and non-invasive cell lines with the Mann-Whitney test revealed a significant correlation for CD63. Grouping of cell lines in relation to threshold values of expression resulted in significant correlations for CD63 (Fisher's exact test p=0.004) and CD151 (p=0.02) but not for CD82 (p=0.065) and CD9 (p=0.168). Expression of CD9, C63 and CD151 was found to be coupled whereas CD82 was expressed independently. This highly significant association points to common mechanisms of gene regulation for this subgroup of tetraspanins. We showed that on basis of absolute amounts of tetraspanin mRNAs, at least in vitro invasiveness is clearly predictable. Our results support the assumption that downregulation of tetraspanins in breast cancer cells is an important step of tumor progression to more malignant phenotypes and underline their important role as mediators in multimolecular membrane protein complexes regulating cell adhesion and migration.

The importance of KAI1 to colorectal cancer (CRC) progression is unclear, with conflicting data regarding changes in KAI1 expression during tumour progression.In situ hybridisation and immunohistochemistry, with a semiquantitative scoring system, were used to assess KAI1 mRNA and protein levels, respectively, in normal colon samples (43), and non-metastatic (24) or metastatic (33) primary cancers, and liver metastases (48).There was significant loss of KAI1 mRNA and protein staining in non-metastatic primary tumours versus normal tissues (Bonferroni, p0.05) but levels recovered to near normal in metastatic primary tumours and liver metastases. Increased KAI1 expression significantly correlated with distant metastases (Mann-Whitney, p = 0.0001, mRNA; p = 0.033, protein), cancer-specific survival (Cox regression analysis p = 0.0002, mRNA; 0.0493, protein) and overall patient survival (p = 0.0001, mRNA). Multivariate analysis (Cox proportional hazards model) confirmed high KAI1 mRNA expression was an independent prognostic indicator of distant metastasis (p0.0001), cancer-specific survival (p0.0001) and overall patient survival (p0.0001).These data indicate a complex relationship between KAI1 and progression of human CRC, in which expression is reduced in localised primary tumours, but regained in disease associated with metastasis.

The molecular basis for the loss of KAI1 expression in invasive and metastatic tumors and tumor cell lines is not understood. Recently, identification of a sequence with homology to the consensus p53-binding motif in the promoter of the KAI1 metastasis suppressor gene, has led to a proposal that transcriptional regulation by p53 controls expression of KAI1, and that a dramatic down-regulation of KAI1 mRNA levels in invasive tumors and many tumor cell lines, is directly due to loss of p53 function. We have tested this hypothesis by assessing KAI1 mRNA levels in a series of 22 cell lines derived from bladder and prostate cancers, in which we confirmed the p53 gene sequence and characterized the functional status of the endogenous p53 protein. We anticipated that cell lines expressing p53 capable of transactivation should express high levels of KAI1 mRNA compared with cell lines expressing defective p53, or which were p53-null. KAI1 mRNA levels were determined by northern analysis using a full-length KAI1 cDNA probe, and varied widely between cell lines examined. However, there was no association between these levels and p53 status. Furthermore, transfection of representative cell lines with wild-type p53, or exposure to DNA damaging agents, had no effect on KAI1 mRNA levels. Our data suggest that p53 is not a major factor regulating levels of KAI1 mRNA in bladder and prostate cancer cell lines.

In this study, we investigated the expression of the KAI1/CD82 gene in oral squamous cell carcinoma (oral SCC). We studied 43 oral SCC patients. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to evaluate expression of this gene, and results were compared to the clinico-pathological findings. Twenty-five specimens (58.1%) were KAI1/CD82-positive, and 18 (41.9%) were negative. There were statistically significant relationships between gene expression and both histological malignancy (P=0.0205) and mode of invasion (P=0.0315). But there were no correlations of expression with tumor status, regional lymph node metastasis, pathological lymph node metastasis or histological differentiation. No significant relationship was observed between patient survival and expression of KAI1/CD82 by tumors. The results of this study suggest that the KAI1/CD82 gene may not be a useful predictor of prognosis, although decreased gene expression may be associated with increased invasive ability of oral SCC.

CD82, a member of tetraspan family (tetraspanin), is a multifunctional molecule that is involved in cell activation, costimulation, and cell spreading of T cells. Here we show that immobilized anti-CD82 monoclonal antibody (mAb) as well as anti-alpha4beta1 integrin mAb induced tyrosine phosphorylation of pp105/Crk-associated substrate lymphocyte type (Cas-L) in human peripheral T cells and H9 cells. Furthermore, one of anti-CD82 mAb (8E4), which induces homotypic aggregation of T cells and H9 cells but has no costimulatory activity, partially inhibited very late antigen (VLA)-4 integrin ligand-mediated costimulation of T cells, whereas it failed to inhibit VLA-5 integrin ligand-mediated costimulation. To further elucidate the relationship between CD82- and VLA-4-mediated signaling pathways we defined the IL-2 production by the costimulation of Jurkat T cells with marginal amount of Cas-L, and subsequently found that mAb against CD82 had strong costimulatory activity to CD3/TCR, whereas mAb against beta1 failed to do so in those cells. We have further demonstrated that this discrepancy between beta1 integrin- and CD82-mediated costimulation partly lies in differential activation of NF-AT, AP-1, and NF-kappaB in Jurkat T cells. In this study, although some functional overlap exists, we provide evidence for distinctive signaling of CD82- and beta1 integrin-mediated costimulation at the transcriptional level of IL-2 gene.

KAI1 is a metastasis suppressor gene located on human chromosome 11p11.2. It belongs to a structurally distinct family of cell surface glycoproteins. Decreased KAI1 expression has been observed in several common solid epithelial tumors, including prostatic, pancreatic, lung, hepatic, colorectal, ovarian, and esophageal cancers. A recent study also observed frequent loss of KAI1 expression in a number of squamous cell carcinomas of the cervix by immunohistochemical technique. To further confirm whether this gene is altered in this malignancy, we analyzed KAI1 expression in various stages of cervical carcinoma by a molecular method. Total cellular RNA was extracted from 84 primary invasive cervical carcinomas and 6 metastatic or recurrent lesions. cDNA was synthesized and was used for real-time quantitative polymerase chain reaction analysis. The level of KAI1 expression was obtained as the value of threshold cycle (Ct) and was quantitated with a comparative Ct method. In addition, paraffin blocks of the tumors were selected and prepared for immunohistochemical study with an anti-KAI1 polyclonal antibody, C-16. Both the real-time quantitative polymerase chain reaction method and immunohistochemical study revealed a frequent decrease in KAI1 expression in invasive cervical cancers and metastatic or recurrent lesions. However, the reduction in KAI1 was not related to progression of the disease. When tumor cell differentiation was analyzed, poorly differentiated tumors showed a greater decrease in KAI1 expression than well or moderately differentiated tumors (P < 0.001). Histologically, KAI1 loss was observed equally in both squamous cell carcinoma and adeno-/adenosquamous carcinoma. Since down-regulation of KAI1 occurs in both early and late stages of cervical cancer, we suggest that its involvement in the progression of this malignancy is an early event.

KAI1 was originally identified in prostate cancer as a metastasis suppressor gene. Recent studies have shown a frequent down-regulation of KAI1 expression in many tumor types, whereas mutation or hypermethylation of the gene is infrequent. The aim of the present study was to examine whether loss of KAI1 expression that might be caused by genetic or epigenetic alterations could contribute to oral carcinogenesis.We analyzed mutational and methylation status of the KAI1 gene and both the mRNA and protein level in a series of oral tumors [28 precancerous lesions, 101 primary oral squamous cell carcinomas (OSCCs), and 30 metastatic OSCCs] and OSCC-derived cell lines. We also examined p53 protein expression, which has been reported to be a candidate activator for the KAI1 gene.With the exception of three microsatellite instabilities in the KAI1 gene, we found no mutations in the coding sequence of the KAI1 gene, no loss of heterozygosity, and no hypermethylation of the KAI1 promoter region in all samples investigated. By immunohistochemistry, however, high frequencies of KAI1 down-regulation were evident not only in the metastatic OSCCs [29 of 30 (97%)] but also in the primary OSCCs [83 of 101 (82%)] and in the precancerous lesions [13 of 28 (46%)]. There was a significant relationship between down-regulation of KAI1 protein expression and primary tumors associated with lymph node metastases (P = 0.0115), whereas there was no statistical correlation between p53 status and KAI1 expression. Taken together, reverse transcription-PCR data were consistent with the protein expression status in 16 patients from whom mRNA was available.Our data suggest that whereas loss of KAI1 protein expression is associated with primary tumors with lymph node metastases, the down-regulation of KAI1 is an early event in the progression of human oral cancer. The down-regulation of KAI1 is not associated with either mutation, allelic loss, methylation of the promoter, or p53 regulation.

Graft-versus-host disease (GVHD), a major complication after allogeneic transplantation, can be abrogated by the Campath (anti-CD52) monoclonal antibody. The induction of acute GVHD requires host antigens to be presented to donor T cells by antigen-presenting cells (APCs). Recent evidence has suggested that only host APCs can interact with donor T cells in the induction of GVHD. Because CD52 has been reported to be expressed on DCs, we reasoned that pretransplant Campath-1G might have a direct effect on circulating DCs in addition to any effects on donor T cells. Using direct immunostaining, we demonstrated expression of CD52 on DCs and that Campath-1G killed purified DCs in vitro. In vivo Campath also depleted DCs. Twenty-four hours after the first dose of Campath-1G, circulating DCs were reduced by a mean of 79% (range, 44%-96%). By day 0 after 5 doses of Campath-1G and chemoradiotherapy conditioning, DCs became undetectable in 7 of 9 cases, whereas in 6 of 7 patients receiving conditioning therapy without Campath-1G, host DCs were still detectable. The reconstitution of circulating DCs after transplantation was not affected by Campath-1G and in both groups DC1 (CD11c+) recovered more rapidly than DC2 (CD11c−). Analysis of chimerism confirmed that the DCs recovering after transplantation in patients receiving Campath-1G were of donor origin. We conclude that in vivo Campath-1G causes a rapid depletion of host circulating DCs and that this may, in part, explain the low incidence of acute GVHD. The reconstitution of donor DCs was not delayed, which may be important in preserving immune reconstitution.

Since its identification as a suppressor gene for prostate cancer metastasis, down-regulation of KAI1/CD82 in a variety of malignancies has been reported.Using immunohistochemistry, we examined KAI1/CD82 expression in surgical specimens obtained from 70 patients with advanced colorectal cancer and its correlation with clinicopathological factors, to clarify their prognostic significance.KAI1/CD82 expression was positive in 55% of the 70 colorectal cancers. There were statistically significant correlations between KAI1/CD82 expression and Dukes' stage, venous invasion, lymph node metastasis, tumor differentiation and liver metastasis. The significant correlation between KAI1/CD82 expression and outcome among patients with Dukes' C cancer (p=0.024) is particularly noteworthy. On multivariate analysis, KAI1/CD82 expression and Dukes' stage were identified as significant and independent prognostic factors (p=0.006 and 0.045, respectively).KAI1/CD82 expression closely correlates with clinicopathological factors for colorectal cancers. KAI1/CD82 expression appears to be a useful prognostic marker.

Recently, we established that a murine monoclonal antibody (MAb) MH8-4 inhibits the motility of the colon cancer cell line RPMI4788 and that it recognizes integrin alpha3. In addition, we have also cloned the motility-related protein-1 (MRP-1)/cluster of differentiation 9 (CD9) as a metastasis suppressor molecule. We investigated integrin alpha3 expression in 114 resected colon cancers using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to evaluate whether these experimental results are of relevance in the prognosis of actual colon cancers. Furthermore, we investigated the correlation of integrin alpha3 with MRP-1/CD9 and KAI1/CD82. Sixty patients (52.6%) were evaluated as integrin alpha3-positive and 54 patients (47.4%) as integrin alpha3-negative. Integrin alpha3 expression was associated with tumor status, lymph node status and pathologic stage. The overall and disease-free survival rates for patients whose tumors were positive for integrin alpha3 were significantly higher than for those with integrin alpha3-negative tumors (p0.001 and p0.001, respectively). This same tendency was observed in node-negative patients (p = 0.007 and p = 0.001, respectively). Integrin alpha3 was found to be the significant prognostic factor in a multivariate analysis using the Cox proportional hazards model (p = 0.036). A correlation was found between integrin alpha3 with MRP-1/CD9 and KAI1/CD82 for stage I tumors. However, no correlation was found in stage III tumors. Our data seem to suggest that low expression of integrin alpha3 is a useful indicator of a poor prognosis for colon cancer patients and that colon cancer progresses following collapse of the complex formed by integrin alpha3 with MRP-1/CD9 and KAI1/CD82.

To investigate the correlation of a newly-defined tumor metastasis suppressor gene KAI1/CD82 and metastatic potential of human cancer cell lines.Expressions of KAI1/CD82 in eight human cancer cell lines of prostate, lung and melanoma with different metastasis potential were studied by Northern blot hybridization and the mutation were detected by PCR-SSCP.Expressions of KAI1 mRNA decreased in human prostate cancer cell lines PC3 and PC3M, and lung cancer cell line PG which were metastatic cancer cell lines (integral light density values: 0.0319, 0.0266 and 0.0549, respectively). Expression of KAI1 mRNA showed high level in PAa, a human lung cancer cell line with no metastasis (integral light density value: 0.7313). In human melanoma cell lines with different metastasis potential (WM35, WM1341b, WM983a and WM451), expression of KAI1 showed moderate expression (integral light density values are 0.1798, 0.1582, 0.1501, 0.1800 respectively). PCR-SSCP analysis indicated the existence of band shift in exon 7 of KAI1 gene in PC3, PG and PC3M.Decreased KAI1/CD82 expression may be associated with enhanced metastatic potential in prostate cancer cell lines and lung cancer cell line other than melanoma cell lines. The decreased expression may be partially nelaled with mutation of KAI1 gene.

KAI1, which is identical to CD82, was initially identified as a metastasis-suppressor gene for human prostate cancer, and its expression is reported to be a favorable prognostic factor for operable human lung cancer. In this study, we examined the functional role of KAI1/CD82 in the late phase of metastatic spread of human lung-cancer cells. For this, KAI1/CD82 cDNA was introduced into KAI1/CD82 low-expressing human lung-cancer cell lines, SBC-3 and PC-14, and then the metastatic potential of the transformants was analyzed by i.v. inoculation of KAI1/CD82-transduced cells, SBC-3/KAI1 and PC-14/KAI1, into NK cell-depleted SCID mice. Contrary to our expectations, KAI1/CD82 gene transfer promoted multiorgan metastasis of i.v.-inoculated human lung-cancer cells, while s.c. tumor growth was unaffected. Cancer cells from metastatic tumors of NK cell-depleted SCID mice injected i.v. with SBC-3/KAI1 expressed appreciable cell-surface KAI1/CD82, and cells not expressing KAI1/CD82 (revertants) were not detected in the tumors. Our findings indicate that under conditions where the host's natural cytotoxicity is suppressed, KAI1/CD82 may enhance the formation of tumors by circulating lung-cancer cells at metastatic sites.

KAI1 is a metastasis suppressor gene for human prostate cancer and is also involved in the progression of a variety of other human cancers. Previously, we have demonstrated that KAI1 expression was down-regulated in metastatic breast cancer cell lines as well as in highly aggressive breast cancer specimens. To determine whether KAI1 expression is responsible for the metastasis suppression in breast cancer, we transfected the human KAI1 cDNA into two highly malignant breast cancer cell lines, LCC6 and MDA-MB-231, which both have low levels of endogenous KAI1 expression. Parental, vector-only transfectants and KAI1 transfectant clones were injected into the mammary fat pads and tail veins, respectively, of athymic nude mice and assessed for both spontaneous and experimental lung metastasis. High KAI1 expression significantly suppressed the metastatic potential of KAI1-transfected LCC6 cells. Metastasis suppression correlated with the reduced rate of tumor growth and a decreased clonogenicity in soft agar. Furthermore, KAI1 expression significantly suppressed the in vitro cell invasion in KAI1-transfected MDA-MB-231 cells. Our results suggested that KAI1 may function as a negative regulator of breast cancer metastasis.

To evaluate KAI1/CD82 expression in Chinese patients with benign prostatic hyperplasia (BPH) and late-stage carcinoma of prostate (CaP).Thirty Chinese patients with benign prostatic hyperplasia and 34 with CaP (adenocarcinoma clinical stage C and D) were analyzed by means of immunohistochemical methods.The KAI1/CD82 expression in BPH tissue was all positive, which was uniformly located on the glandular cell membrane at the cell-to-cell borders, but KAI1/CD82 expression in metastasis CaP tissues was either significantly lower than that of BPH or negative, and the immunostaining pattern was not continuous. In late-stage CaP KAI1/CD82 expression was correlated inversely to the pathological grade ( P0.05), but not to clinical stage ( P0.05).The authors believe that decreased and negative KAI1/CD82 expression in late-stage CaP may be related to tumor progression and metastasis, and appears to be a prognostic marker.

Loss of KAI1 protein is a frequent event in various types of cancer and has been reported to be associated with increased rate of metastasis and shorter survival time.Expression of KAI1 and p53 was immunohistochemically determined in 75 biopsy specimens from patients with primary irradiated, invasive squamous cell cervical cancer and 30 cases with CIN I-III.In invasive cancer, 22 cases showed strong expression, 42 cases down-regulation, and 11 cases no expression of KAI1. All specimens with CIN I showed strong KAI1 expression, while in CIN II and III down-regulation was observed in 45% of cases. There was no significant correlation of KAI1 expression with clinical and histopathological parameters. No influence of KAI1 expression on prognosis was observed.KAI1-down-regulation is a frequent event in cervical cancer occurring early in carcinogenesis. KAI1 expression did not influence the prognosis of patients with primary irradiated cervical cancer.

KAI1/CD82 has been shown to be a metastasis suppressor for several human cancers, and a recent study revealed that wild-type tumor suppressor p53 can directly activate KAI1/CD82 gene expression. However, the response of KAI1/CD82 expression in cancer cells to exogenous stimulants has not been investigated. The present study examined whether tumor necrosis factor (TNF), which mediates many of the cellular responses associated with inflammatory reactions or cancer progression, can affect the KAI1/CD82 expression in lung cancer cells and, if so, whether nuclear factor (NF)-kappaB, a key molecule in TNF-mediated gene expression, is involved in the mechanism of KAI1/CD82 induction. Our results demonstrated that expression of KAI1/CD82 in PC-14 cells expressing mutant p53 could be augmented by TNF-alpha, and that transfer of the gene for a specific inhibitor of NF-kappaB, IkappaB alphaSR (mutant IkappaB alpha; NF-kappaB super-repressor), into PC-14 cells could inhibit this augmentation. The amount of NF-kappaB in the nucleus of PC-14/IkappaB alphaSR cells correlated well with KAI1/CD82 mRNA and protein expression. In addition, IkappaB alphaSR gene transfer inhibited the spontaneous expression of KAI1/CD82 protein in KAI1/CD82-high-expressing RERF-LC-OK cells, which contain a mutant-type p53. These observations indicate that NF-kappaB activation may play a role in the regulation of KAI1/CD82 expression in lung cancer cells independently of wild-type p53, and suggest that KAI1/CD82 expression may be regulated by interaction with the host microenvironment.

Laminin (LN)- or fibronectin (FN)-dependent adhesion in Krieger's ldlD 14 (D14) cells is enhanced significantly in the presence vs absence, of galactose (Gal), whereas LN- or FN-induced haptotactic cell motility is barely affected unless cells express CD82 by its gene transfection (cells termed D14/CD82). The effect of CD82 on LN- or FN-induced motility is based on its ability to associate with alpha3 or alpha5 integrin to form a complex associated with a low-density lipid membrane domain (termed GEM or GSD). Complex formation is greatly affected by N-glycosylation of both integrin and CD82, as well as by concurrent GM3 ganglioside synthesis. The effect of glycosylation on alpha5-CD82 complex was also studied in D14 cells expressing mutant CD82, defective in all three N-glycosylation sites. LN-induced motility was greatly inhibited, whereas FN-induced motility was enhanced, with complete N-glycosylation in D14/CD82 cells in Gal-added medium, whereby alpha5-CD82 complex formation did not occur or occurred at a minimal level. Both LN- and FN-induced motility were inhibited when N-glycosylation was impaired, or N-glycosylation of CD82 was deleted, whereby alpha5-CD82 complex formation occurred strongly. Thus, glycosylation profoundly affects interaction of integrin with CD82, leading to significant inhibition or promotion of cell motility.

The present is an overview of recent data that describes the genetic underpinnings of the suppression of cancer metastasis. Despite the explosion of new information about the genetics of cancer, only six human genes have thus far been shown to suppress metastasis functionally. Not all have been shown to be functional in breast carcinoma. Several additional genes inhibit various steps of the metastatic cascade, but do not necessarily block metastasis when tested using in vivo assays. The implications of this are discussed. Two recently discovered metastasis suppressor genes block proliferation of tumor cells at a secondary site, offering a new target for therapeutic intervention.

We have previously shown that levels of KAI1 mRNA are dramatically reduced in invasive human bladder cancers. To further investigate the role of KAI1 in bladder cancer, we have examined the relationship between KAI1 mRNA levels and cell behaviour in 18 bladder cancer cell lines and a virus-transformed uro-epithelial cell line. We found that low KAI1 mRNA levels correlated with increased in vitro invasive ability, reduced Ca 2+ -dependent and -independent cell–cell adhesion and reduced adhesion to fibronectin. These data support the idea that loss of KAI1 expression is an important factor in tumour cell invasive behaviour.

Expression of a newly described inhibitor of tumour metastasis, KAI1, was examined in bladder cancer progression and compared with the expression of p53 and pRb, which are markers of advanced disease. KAI1 mRNA (by in situ hybridization) and protein levels (by immunohistochemistry) were examined in 135 paraffin-embedded bladder tissue sections. Significant decreases in KAI1 mRNA and protein levels were detected between normal and tumour tissue (p

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors. The membrane-anchored form of HB-EGF (proHB-EGF) is mitogenically active to neighboring cells as well as being a precursor of the soluble form. In addition to its mitogenic activity, proHB-EGF has the property of binding to diphtheria toxin (DT), serving as the specific receptor for DT. Tetramembrane-spanning protein CD9, a member of the TM4 superfamily, is physically associated with proHB-EGF at the cell surface and up-regulates both mitogenic and DT binding activities of proHB-EGF. To understand this up-regulation mechanism, we studied essential regions of both CD9 and proHB-EGF for up-regulation. Immunoprecipitation experiments revealed that not only CD9 but also other TM4 proteins including CD63, CD81, and CD82 associate with proHB-EGF on the cell surface. However, these TM4 proteins did not up-regulate DT binding activity of proHB-EGF. Transfection of a series of chimeric constructs comprising CD9 and CD81 showed that the major extracellular domain of CD9 is essential for up-regulation. Assays of DT binding activity and juxtacrine mitogenic activity of the deletion mutants of proHB-EGF and chimeric molecules, derived from proHB-EGF and TGF-alpha, showed that the essential domain of proHB-EGF for up-regulation is the EGF-like domain. These results indicate that the interaction of the extracellular domains of both molecules is important for up-regulation.

We previously demonstrated that CD82, expressed on both T cells and antigen-presenting cells (APC), plays an important role as a co-stimulatory molecule especially in the early phase of T cell activation. We also showed that the CD82 expression level is up-regulated on activated T cells and memory T cells. This up-regulation enhances both T cell-T cell and T cell-APC interactions. In this study, we further investigated the mechanism of CD82-mediated cell-cell adhesion. The enhanced adhesion between CD82-overexpressing Jurkat cells was completely blocked by anti-ICAM-1 / LFA-1 monoclonal antibodies. Increased interaction of LFA-1 with ICAM-1 was further confirmed by enhanced adhesion of CD82-overexpressing Jurkat cells to immobilized ICAM-1-Ig. CD82 co-immunoprecipitated with LFA-1 from Jurkat cells and CD82 and LFA-1 colocalized at an adhesion foci. These results suggest that the T cell stimulation via anti-CD3 cross-linking or phorbol myristate acetate treatment up-regulates CD82 expression, leading to the colocalization of CD82 and LFA-1, and results in enhanced interaction between LFA-1 and ICAM-1. In addition, a blocking experiment using monoclonal antibodies suggested that CD82 and LFA-1 molecules on APC are also important for the optimal activation of T cells. This is the first report that describes the enhancement of cell-cell interaction through LFA-1 and ICAM-1 by the overexpression of another cell surface molecule, CD82.

The transmembrane 4 superfamily member KAI1 (CD82) has been shown to inhibit pulmonary metastases in experimental metastasis models of prostate cancer and melanoma. KAI1 expression is decreased in the progression of common solid epithelial tumors of adulthood, including lung, prostate, breast, esophageal, gastric, pancreatic, and bladder cancers. The purpose of our study was to investigate KAI1 expression in the progression of human colorectal cancer. We first analyzed 20 colorectal cancer cell lines by immunoblot techniques. KAI1 was expressed heterogeneously, with the tumor cell lines having a more complex degree of glycosylation compared with that of the normal colonic tissue. KAI1 was highly expressed in the primary SW480 colon cancer cell line but was down-regulated 15-fold in the matched metastatic SW620 cell line. We also investigated KAI1 protein expression by immunohistochemistry in tissues from 84 patients with colorectal cancer. Each tissue section was assigned a KAI1 mean score (KMS) from 0 to 300 based on the product of the percentage of cells that stained for KAI1 and the intensity of the stain (1, 2, or 3). In 84 patients with colorectal cancer, KAI1 was expressed at high levels in normal colonic mucosa (KMS 226) but was expressed at lower levels in the primary tumors (KMS 65; P0.0001). In a subset of 12 patients with stage IV metastatic disease, we observed a progressive down-regulation of KAI1, from the normal adjacent colonic mucosa (KMS 193) to the primary tumor (KMS 72; P = 0.0001) to the liver metastasis (KMS 25; tumor compared with metastasis, P = 0.0135). We found no correlation between loss of KAI1 expression and stage of disease. In 10 patients, we also noted loss of KAI1 expression in the transition from normal colonic mucosa (KMS 237) to adenoma (KMS 174) to carcinoma (KMS 62; P0.0167 for all three comparisons). We conclude that the down-regulation of KAI1 occurs early in the progression of colorectal cancer.

CD82 (KAI1) is a member of the tetraspan transmembrane protein family which has been cloned from lymphoblastoid variant cell lines. However, a role for CD82 in early normal and malignant haemopoiesis has not yet been characterized. We studied the CD82 expression in 33 normal donor samples and 98 leukaemias by fluorescence activated cell sorting (FACS) and reverse transcriptase polymerase chain reaction (RT-PCR). We demonstrated that CD82 was moderately expressed in the vast majority of normal granulocytes and monocytes. In contrast, only about one third of the peripheral blood lymphocytes were weakly CD82 positive (CD82+). Interestingly, judgement of the CD82 transcription and expression in various leukaemias revealed that CD82 was overexpressed in chronic myeloid leukaemia (CML) patients in accelerated or blastic phase (CML-AP/BP) as well as in acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL) patients. Analysis of AML patients with CD34+/CD82+ blasts prompted us to expand our studies on haemopoietic CD34+ progenitor cells. Intriguingly, 84-95% of the CD34+ cells isolated from healthy bone marrow, cord blood or peripheral blood were highly CD82+. CD82 was abundantly expressed on primitive as well as on committed haemopoietic progenitor cells. After in vitro induction of myeloid differentiation in CD34+ peripheral blood progenitor cells (PBPC), the expression of CD82 decreased to levels similar to those found on peripheral blood granulocytes. These observations suggest for the first time a role for CD82 in normal and malignant haemopoiesis.

The KAI1 gene, isolated from human chromosome 11p11.2, has been implicated as a prostate cancer metastasis suppressor gene. Recent studies have demonstrated that the expression of KAI1 protein is reduced in metastases of human prostate cancers and is inversely correlated with tumour grade. The objectives of the present work were to determine whether alterations of KAI1 at a genetic level in localized prostate cancers correlate with degrees of differentiation. This paper reports the application of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern analysis to two different regions of the KAI1 gene on 35 microdissected primary prostate cancer specimens and demonstrates a biphasic pattern of KAI1 expression according to histological grade. KAI1 mRNA, relative to the housekeeping gene beta -actin, was elevated in low-grade primary prostate cancer (2.7+/-0.4) compared with non-malignant (hyperplastic) prostatic tisues (0.92+/-0.02, p0.05), yet reduced in high-grade primary cancers (0.61+/-0.11, p0. 05). These data demonstrate, for the first time, that KAI1 is biphasically expressed in primary prostate cancers and suggest that hyperexpression of KAI1 in low-grade prostate cancer may be associated with restraint of tumour progression, whereas a relative decrease in KAI1 gene expression may accompany more aggressive cancers through loss of such restraint. This differential expression of the metastasis suppressor gene KAI1 in primary prostate cancers may have important prognostic implications for the development of subsequent metastases. Should the level of KAI1 in primary prostate cancer be correlated with patient outcome such information may, in the future, enable more intensive adjuvant therapy to be directed to those patients identified to be at greatest risk of metastasis.

The metastasis suppressor gene KAI1 was identified by its ability to inhibit the formation of pulmonary metastases in experimental models for prostatic carcinoma. Down-regulation of this gene may be correlated with the invasive phenotype in melanomas and colon and bladder carcinomas and with the metastatic phenotype in carcinomas of the lung, breast, prostate, and pancreas. The goal of our study was to establish an immunohistochemical method to detect KAI1 expression in archival tissues. Using cell lines with known KAI1 levels and paraffin-embedded KAI1 positive tissues as controls, we observed strong membrane staining in lymphoid follicular centers and squamous epithelia. We then demonstrated the utility of our assay by studying KAI1 expression in 34 lymphoid and 57 squamous lesions. All eight reactive lymph nodes were KAI1 positive. In contrast, three of 13 follicular small cleaved and five of 13 diffuse large cell lymphomas were KAI1 negative. Seventy-nine percent (37 of 47) of invasive squamous cell carcinomas from the lung (n = 15), head and neck (n = 18), and cervix (n = 14) showed extensive KAI1 down-regulation. Loss of KAI1 expression was also found in a subset of 10 high-grade cervical dysplasias. Our data show that (i) immunohistochemistry is a suitable technique for evaluating KAI1 expression in archival tissues; (ii) KAI1 was not expressed in a subset of both low-grade and high-grade lymphomas; and (iii) there was extensive down-regulation of KAI1 in squamous cell carcinomas, suggestive of an important role of the gene in the suppression of invasion in these malignancies.

Nerve growth factor (NGF) is expressed in the prostate, where it appears to be involved in the control of epithelial cell growth and differentiation. NGF production is decreased in prostate tumors. However, the role of this neurotrophin in the control of proliferation and progression of prostate cancers is still a matter of investigation. Prostate adenocarcinomas are telomerase-positive tumors. Chronic exposure of DU145 and PC3 prostate tumor cell lines to NGF resulted in a dramatic down-regulation of telomerase activity. This effect was correlated in terms of concentrations and time with a remarkable down-regulation of cell proliferation both in vitro and in vivo but was not secondary to NGF-induced quiescence. No down-regulation of telomerase activity was, in fact, detectable during serum starvation-induced quiescence. LNCaP cells, which do not express NGF receptors, appear to be insensitive to the actions of NGF. DU145 and PC3 cells do not express the KAI1 metastasis suppressor gene, which is present in the prostate and is progressively lost during the progression of prostate cancers. Chronic NGF treatment strongly induced the reexpression of this gene in these cell lines, and this effect was correlated with the suppression of their invasive potential in vitro. The data presented here suggest that NGF reverts two metastatic prostate cancer cell lines to slowly proliferating, noninvasive phenotypes characterized by a very low telomerase activity and by the expression of the KAI1 metastasis suppressor gene.

Metastasis-suppressing gene product CD82 and its analogue CD9 are considered to suppress the malignancy of various human cancers, although the rationale for this effect is unknown. The present study addresses phenotypic changes in Chinese hamster ovary mutant cell line ldlD deficient in UDP-Glc 4-epimerase and expressing CD82 or CD9 by cDNA transfection. Only CD82- or CD9-expressing cells grown in Gal-supplemented medium showed reduced motility and massive cell death, which are characteristic of apoptosis, after a latent period. Under this condition, endogenous GM3 synthesis was observed as a common factor, and N-glycosylation occurred at a high level in CD82 and to a lesser extent in CD9. Thus, the malignancy-suppressing effect of CD82 or CD9 is based partially on cell motility inhibition and apoptosis induction promoted by concurrent GM3 synthesis and N-glycosylation.

Although the mechanisms of action of the transmembrane superfamilies, motility-related protein-1 (MRP-1/CD9) and KAI1/CD82, are not well known, they are reported to suppress the metastasis of several kinds of cancers. The suppression of cell motility by MRP-1/CD9 may cause suppression of the metastasis. As we could not find any reports concerning the expression of MRP-1/CD9 and KAI1/CD82 in oesophageal cancers we investigated their expression in oesophageal specimens. We conducted immunohistochemical staining for MRP-1/CD9 against 108 cases of oesophageal squamous cell carcinoma using anti-MRP-1/CD9 monoclonal antibody M31-15, and for KAI1/CD82 against 104 cases using anti-KAI1/CD82 monoclonal antibody C33. To investigate the gradual expression of MRP-1/CD9 and KAI1/CD82, 24 oesophageal dysplasias were immunohistochemically stained using the same method and then investigated. The expression of both MRP-1/CD9 and KAI1/CD82 were positive on the cell membranes of normal oesophageal epithelial cells, but reduced or negative in the cancer cells. Reduced MRP-1/CD9 expressions significantly correlated to tumour depth (P = 0.0009). We found a significantly greater number of reduced or negative expression of MRP-1/CD9 and KAI1/CD82 in lymph node metastatic cases (P = 0.0003 and P = 0.0129, respectively), but not in distant metastatic cases. The 5-year survival rate of MRP-1/CD9-negative and reduced patients was significantly worse than those of positive patients (n = 108, curative cases, RO). Few cases remained KAI1/CD82-positive (9.6%; 10/104) in oesophageal cancer. Twenty (83.3%) and twenty-two (91.7%) cases out of 24 dysplasias were defined as KAI1/CD82-positive and MRP-1/CD9-positive, respectively. The decrease in MRP-1/CD9 and KAI1/CD82 expression may facilitate lymph node metastasis in oesophageal squamous cell carcinomas. Knowing the status of the expression of MRP-1/CD9 appears helpful in predicting the prognosis for each patient. © 1999 Cancer Research Campaign

KAI1 gene is a novel metastasis suppressor gene. Its transcription level has been reported to be related with metastases in several kinds of tumors. The Authors used RT-PCR method to investigate the expression levels of KAI1 gene in hepatocellular carcinoma (HCC) tissues from 42 patients. The results demonstrated that 57.1% of tumor tissues were positive in KAI1 expression. There was no statistically significant difference on KAI1 gene expression rate between large (5 cm) and small (=5 cm) tumors, encapsulated and non-capsulated tumors, as well as alpha-fetoprotein (AFP) positive (20 ug/ml) and negative (=20 ug/ml) tumors; however, the expression rate was higher in tumors without intrahepatic metastasis than in those with intrahepatic metastasis (63.9% vs. 16.7%, P0.05). It is suggested that KAI1 gene expression may be related to the metastases of hepatocellular carcinoma.

In this report, we provide new evidence of a crosstalk between T cell activation and adhesion processes through a functional cytokeleton. We show that CD82 signaling induces long-lasting adhesion, spreading and development of membrane extensions, involving actin polymerization. Addition of various co-stimuli (phorbol 12-myristate 13-acetate or monoclonal antibodies to CD3 or CD2) increases the CD82-induced morphological alterations and, reciprocally, CD82 engagement synergizes with these stimuli to induce T cell activation as indicated by both primary tyrosine phosphorylation and IL-2 production. Different kinases are involved in both processes. CD82 co-signaling involves src kinases including p56 Ick. On the other hand, the CD82-induced alterations of cell morphology are negatively regulated by cAMP-dependent kinases independently of activation of src kinases. Simultaneously with cytoskeletal rearrangements, we observed an inducible association of CD82 with the cytoskeletal matrix. In addition, the potentiating and stabilizing effects induced by CD82 cross-linking on tyrosine phosphorylation were abolished by cytoskeleton-disrupting agents. These results suggest that the actin polymerization triggered by CD82, through its ability to associate with the cytoskeletal matrix, is the primary step involved in the CD82 induced co-stimulatory activity. Our data provide further evidence for a direct role of the actin cytoskeleton as a major component for sustained signal transduction in T cells and suggest that tetraspanins could be "membrane organizers" connecting both surface and intracellular molecules.

We previously isolated the human KAI1 gene encoding a transmembrane protein which suppresses metastatic ability in Dunning R3327 AT6.1 rat prostate cancer cells when transfected into these cells. The AT6.1 subline is one of the more aggressive sublines among the Dunning R-3327 system of rat prostate cancers. This raises the issue of whether downregulation of KAI1 expression consistently occurs during the acquisition of high metastatic ability by members of the Dunning system of rat prostate cancers.To investigate this possibility, the rat homologue of the KAI1gene was identified, using a combination of cDNA library screening and 5'-RACE and DNA sequencing. Based on this information, a rat-specific cDNA probe was developed and used for Northern blot analysis of KAI1 expression in normal rat tissues and a series of sublines of Dunning R3327 cells that vary widely in their metastatic abilities.The rat KAI1 gene encoded a protein of 266 amino acids which has 77% identity to the human KAI1 protein. In normal tissues, KAI1 is expressed predominantly as a 2.0-kb-sized transcript. Several tissues (e.g., skeletal muscle and prostate) also express a minor 1.8-kb-sized RNA. Northern blot analysis of a series of Dunning sublines demonstrated that all sublines expressed both the 2.0- and 1.8-kb KAI1 RNA transcripts. However, quantitative levels of the 2.0- vs. 1.8-kb KAI1 RNA were variable among sublines. Downregulation of expression of the 2.0-kb KAI1 transcript was statistically correlated with the acquisition of high metastatic ability within this system of prostate cancer sublines. In contrast, the 1.8-kb transcript was upregulated in all of the more aggressive sublines, but this enhanced expression was not specifically correlated with metastatic ability.These studies demonstrated that downregulation of the 2.0-kb KAI1 mRNA is associated with the acquisition of high metastatic ability by prostate cancer cells.

Down-regulation of KAI1 expression has been shown to be associated with formation of metastases or disease progression in prostate and pancreatic cancer. In the present study we analyzed the expression pattern of KAI1 in metastatic and nonmetastatic hepatocellular carcinomas (HCCs) in comparison with normal livers to evaluate whether alteration of KAI1 also facilitates the metastatic ability in this malignancy. Thirty-nine primary HCCs and 10 normal liver tissue samples were studied for KAI1 messenger RNA (mRNA) expression with use of Northern blot analysis and in situ hybridization. By Northern blot analysis, moderate to strong KAI1 mRNA expression was present in normal liver samples. In contrast, KAI1 mRNA expression in tissue samples of primary HCCs was markedly decreased compared with normal controls. The normal/tumor ratio of KAI1 mRNA expression was 2.6:1 (P.01). Primary HCCs that gave rise to metastasis showed significantly lower KAI1 mRNA levels than nonmetastasized HCCs (P. 05). As seen by in situ hybridization, moderate to strong cytoplasmic KAI1 mRNA staining was present in almost all normal hepatocytes. Bile ducts, blood vessels, and connective tissue showed no or only faint KAI1 mRNA expression in the normal liver samples. In nonmetastatic HCCs, the cancer cells exhibited in situ hybridization signals that were similar to the normal controls. In contrast, most of the primary HCC cells in samples with metastases showed only faint or moderate KAI1 mRNA expression predominantly in the perinuclear regions. When KAI1 mRNA expression of primary hepatocellular cancer cells was compared with metastasized cancer cells in lymph nodes, with intrahepatic satellite metastasis, or with peritoneal metastasis in the same patients, significantly lower (P.01) KAI1 mRNA levels were present in the metastasized HCC cells. Reduced KAI1 mRNA in HCC cells seems to influence their metastatic ability and thereby enhances the malignant potential of HCC.

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.

MRP-1/CD9 , KAI1/CD82 , and ME491/CD63 , have been reported to be associated with the metastatic potential of solid tumors. The aim of this study was to determine whether their expression in tumor tissues is a useful indicator for prognosis in breast cancer patients. We studied 109 breast cancer patients who underwent surgery. Quantitative reverse transcription-polymerase chain reaction analysis was performed to evaluate the expression of these genes. The results were confirmed with immunohistochemistry. All of the carcinomas were ME491/CD63 positive. Thirty-six tumors were MRP-1/CD9 negative. The disease-free survival rate and the 5-year survival rate of patients with MRP-1/CD9 -negative tumors were both significantly lower than that in patients with MRP-1/CD9 -positive tumors ( P = 0.0005 and P = 0.0380, respectively). Sixty-five tumors were KAI1/CD82 negative. The disease-free survival rate of patients with KAI1/CD82 -negative tumors was significantly lower than that of patients with KAI1/CD82 -positive tumors ( P = 0.0065). Cox regression analysis demonstrated that MRP-1/CD9 status ( P = 0.0016) and KAI1/CD82 status ( P = 0.0234) were useful indicators for the disease-free survival of breast cancer patients. The disease-free survival rate and 5-year survival rate of patients with either MRP-1/CD9 -negative or KAI1/CD82 -negative tumors were both significantly lower than patients who were positive for both genes ( P = 0.0003 and P = 0.0292, respectively). The expression of MRP-1/CD9 and KAI1/CD82 genes are useful indicators of a poor prognosis in breast cancer patients.

KAI1 belongs to a structurally distinct family of membrane glycoproteins, which function via cell-cell and cell-extracellular matrix interactions, thereby potentially influencing the ability of cancer cells to invade tissues and to metastasize into lymph nodes and distant organs. In the present study, we examined KAI1 expression in lymph node and liver metastases in comparison with primary pancreatic cancer to evaluate its influence on metastasis. KAI1 mRNA analysis was performed by Northern blot analysis and in situ hybridization. In addition, the respective protein was studied by immunostaining. Fourteen primary pancreatic cancer samples in which no lymph node metastases were present and 25 primary pancreatic cancer samples in which lymph node metastases were present at the time of tumor resection were included. In 20 of these cases, primary pancreatic cancer tissues and corresponding lymph node metastases from the same patient were studied. Furthermore, 11 liver metastases were available for KAI1 analysis. Increased steady-state levels of KAI1 mRNA were found in 33/39 (85%) primary pancreatic cancers in comparison with normal controls. Statistical analysis of KAI1 mRNA levels and clinical parameters of the patients revealed that KAI1 mRNA levels were significantly higher in non-metastasized tumors compared with tumors in which lymph node or distant metastases were present. In lymph node metastases KAI1 mRNA expression was lower than in the corresponding primary tumors: In 14 of 20 lymph node metastases no KAI1 mRNA expression and in 6 of 20 lymph node metastases only weak KAI1 mRNA levels were present in some cancer cells. Cancer cells of distant metastases were devoid of or exhibited low KAI1 mRNA levels compared with those of primary pancreatic cancers. A similar pattern was observed by immunostaining. These data support the hypothesis that KAI1 gene expression might influence the metastatic ability of pancreatic cancer cells in vivo. Reduction of KAI1 appears to promote cancer cell spread in lymph nodes and distant organs.

BACKGROUND The KAI1/CD82 gene, the product of which is a member of the transmembrane-4 superfamily, is a suppressor of metastasis; as a result, it is inversely associated with tumor progression and is a favorable prognostic factor in some tumors. This study was performed to determine the prognostic value of KAI1/CD82 protein levels in nonsmall cell lung carcinoma (NSCLC). In addition, levels of KAI1/CD82 expression in metastatic lesions were determined and compared with those in primary NSCLC lesions. METHODS KAI1/CD82 expression in 200 NSCLC patients who underwent potentially curative surgery was immunohistochemically detected with C33, an anti-KAI1/CD82 monoclonal antibody. According to the degree of KAI1/CD82 positive cancer cells within the tumor tissue, each sample was classified as KAI1/CD82 positive, KAI1/CD82 reduced, or KAI1/CD82 negative. RESULTS Sixty-five samples (32.5%) were KAI1/CD82 positive, 31 (15.5%) were reduced, and 104 (52%) were negative. There was no significant association between KAI1/CD82 expression and clinicopathologic factors, but patients who were positive for KAI1/CD82 expression had significantly favorable prognoses for overall survival (P = 0.0026) and disease free survival (DFS; P = 0.0007) compared with the other groups. In particular, among patients with adenocarcinoma, similar results were even more significant. In multivariate analysis, immunohistochemical KAI1/CD82 expression in patients with NSCLC was an independent prognostic factor for overall survival and DFS; in those with adenocarcinoma, it was an even more valuable factor. In some patients with NSCLC, especially those with adenocarcinoma, KAI1/CD82 expression levels in metastatic lesions were diminished compared with levels of expression in the primary lung lesions. CONCLUSIONS The immunohistochemically determined level of KAI1/CD82 expression in NSCLC cells within tumor tissue appears to be a favorable prognostic factor for overall survival as well as DFS. The results of this study suggest that decreased KAI1/CD82 expression may be associated with tumor progression and enhanced metastatic potential in some patients with this disease. Cancer 1998;83:466-474. © 1998 American Cancer Society.

The KAI1 gene maps to chromosome 11p11.2, is a metastasis-suppressor gene for human prostate cancer and also is involved in the progression of human pancreatic and non-small cell lung cancer. Recently, we showed that introduction of a single copy of normal, neomycin-tagged human chromosome 11 into highly metastatic MDA-MB-435 breast cancer cells suppressed breast cancer metastasis. Concomitantly, KAI 1 levels were higher in chromosome 11/MDA-MB-435 cell clones. The purpose of this study was to test whether KAI 1 expression is indicative of breast cancer metastasis using a panel of immortalized breast epithelial and breast cancer cell lines that represent multiple stages of breast cancer progression. Metastatic cell clones isolated from the parental mixed, wild-type population of MDA-MB-435 cells expressed the lowest levels of KAI 1 mRNA and chromosome 11 containing MDA-MB-435 (neol1/MDA-MB-435.A3 and neol1/MDA-MB-435.B1) cells had approximately twice as much KAI 1 mRNA than the parental clones. MCF-10A, an immortalized normal-like non-tumorigenic mammary epithelial cell line, had the highest level of KAI 1 mRNA. We compared the metastatic propensity and invasive ability of a continuum of breast cancer cells with varying degrees of progression toward malignancy and found that these parameters tended to correlate inversely with KAI 1 mRNA expression. These data suggest that, in addition to its role in human prostate, pancreatic and non-small cell lung cancer, KAI 1 may also be a useful marker for staging human breast disease.

PURPOSE The transmembrane-4 superfamily (TM4SF) is a recently discovered family of genes. Of the TM4SF members, MRP-1/CD9, KAI1/CD82, and ME491/CD63 have been reported to modulate tumor progression or metastasis. In this study, we investigated the relationships between these three genes, MRP-1, KAI1, and ME491, in patients with non-small-cell lung cancers (NSCLCs). Moreover, we assessed the prognostic value of evaluating the expressions of MRP-1, KAI1, and ME491 simultaneously in NSCLCs. PATIENTS AND METHODS One hundred seventy-two patients up to stage IIIB NSCLC underwent radical surgery during the period of January 1991 through June 1994. Using a quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, we studied the expression of MRP-1, KAI1, and ME491 genes in these patients. RESULTS We found that 109 patients (63.4%) had MRP-1-positive tumors and 42 patients (24.4%) had KAl1-positive tumors. Conversely, all 172 patients expressed ME491. No relationship was found between MRP-1 expression and KAI1 expression. We classified these patients into three groups. The 36 patients who were positive for both MRP-1 and KAI1 were defined as group A; the 79 patients with reduced expression of either MRP-1 or KAI1 were defined as group B, and the remaining 57 patients with reduced expression of both MRP-1 and KAI1 were defined as group C. This new classification was correlated with nodal status, tumor status, and pathologic stage (P = .0056, P = .0003, and P < .0001, respectively). In NSCLC patients, the 5-year survival rate of group A patients was significantly better than that of group B patients and much better than that of group C patients (86.8%, 53.9%, and 31.5%, respectively; P < .0001). Cox multivariate regression analysis showed that this new classification in NSCLCs was a significant prognostic factor, as was the nodal status (P < .0001). CONCLUSION Our results suggest that a low MRP-1 and KAI1 expression by tumors of the lung may be associated with poor prognosis. It is conceivable that the evaluation for MRP-1 and KAI1 expression may identify node-negative lung cancer patients who are at high risk for early disease recurrence, and thus need intensive adjuvant therapy.

KAI1 is a potential metastatic suppressor gene for prostate cancer. We found by Northern blot analysis that six of ten (60%) gastric and colon cancer cell lines exhibited undetectable or very low expression level of KAI1 mRNA. The effects of KAI1 on the adhesion, motility and invasiveness of colon cancer cells was therefore investigated by using two kinds of stable transfectants, i.e., antisense transfectants of BM314 cells whose KAI1 mRNA expression was suppressed by transfer of antisense KAI1 cDNA and sense transfectants of DLD-1 cells with the enhanced KAI1 mRNA by sense cDNA transfer. The following results were obtained: (1) KAI1 gene expression had no significant effect on in vitro cell growth rate of colon cancer BM314 and DLD-1 cells; (2) Cell aggregation assay showed that KAI1 enhanced the Ca++-independent aggregatability of those colon cancer cells; (3) It was revealed by cell motility and invasion assays that KAI1 suppressed both the motility and in vitro invasiveness of those cells and (4) Furthermore, both the binding to fibronectin and the migration on fibronectin-coated plates of those cells were inhibited by KAI1 expression. These suggest that reduced KAI1 gene expression may contribute to the invasiveness and metastatic ability of colon cancer cells.

Molecules of the tetraspan superfamily are engaged in multimolecular complexes containing other proteins such as β1 integrins and MHC antigens. Although their functions are not clear, they have been suggested to play a role in cell adhesion and migration, signal transduction, and costimulation We have in this paper directly compared the functional properties of four tetraspans, CD9, CD53, CD81, and CD82. mAbs to any of these molecules were able to deliver a costimulatory signal for CD3-mediated activation of the T cell line Jurkat. CD82 mAbs were the most efficient in triggering this effect. Moreover, engagement of CD9, CD81, and CD82 induced the homotypic agregation of the megakaryocytic cell line HEL, and inhibited the migration of this cell line. Similar results were obtained with the preB cell line NALM-6 using the CD9 and CD81 mAbs. The CD81 mAb 5A6 produced the strongest effects. Therefore, the tetraspans are recognized by mAbs which produce similar effects on the same cell lines. This is consistent with the tetraspans being included in large molecular complexes and possibly forming a tetraspan network (the tetraspan web). We also demonstrate that the tetraspans are likely to keep specific functional properties inside this network. Indeed, we have demonstrated that the human CD9 is able, like the monkey molecule, to upregulate the activity of the transmembrane precursor of heparin-binding EGF as a receptor for the diphtheria toxin when cotransfected in murine LM cells. Neither CD81, nor CD82 had such activity. By using chimeric CD9/CD81 molecules we demonstrate that this activity requires the second half of CD9, which contains the large extracellular loop, the fourth transmembrane region, and the last short cytoplasmic domain.

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.

Tumor cell metastasis to distant organs is an inefficient process that is limited in part by recently identified metastasis suppressors. Interactions between tumor cells and the surrounding stroma are thought to control much of cancer progression. In the August issue of Nature Medicine, Bandyopadhyay et al. (2006) demonstrate that specific cell surface interactions between the metastasis suppressor KAI1 on tumor cells and the decoy cytokine receptor DARC on adjacent vascular cells triggers senescence in the tumor cells and suppresses metastasis. These new observations demonstrate how metastasis suppressors can relay the restraint imposed by the stroma onto disseminating tumor cells.

BACKGROUND We recently isolated the KAI1 gene, a metastasis suppressor gene for prostate cancer, from human chromosome region 11p13–cen-containing rat prostate cancer cells. The present study was performed to further locate the region of the KAI1 gene on the short arm of chromosome 11, and to examine whether loss of this region is significant during progression of human prostate cancer. METHODS The small portion of human chromosome 11 (i.e., 11p13-cen) was reintroduced into highly metastatic rat prostate cancer cells by using microcell-mediated chromosome transfer. Loss of heterozygosity (LOH) at polymorphic microsatellite loci on the human chromosome 11 was examined in human prostate cancer tissues. RESULTS The minimum region of human chromosome 11 that contained the KAI1 gene was located on the proximal region of 11p11.2 divided by the D11S554 locus. The percentage of LOH or allelic imbalance at the D11S1344 locus, which is located on the same region as the KAI1 locus, in metastasis tissues from autopsy cases who died from metastatic prostate cancer was 70% (7 of 10 informative cases), whereas the percentages in primary tumors from the same cases and from cases with clinically localized prostate cancer were 33% (3 of 9 informative cases) and 8% (1 of 12 informative cases), respectively. CONCLUSIONS These findings demonstrate a high frequency of LOH or allelic imbalance at the centromeric region of 11p, which contains the KAI1 gene in advanced prostate cancer. Prostate 32:205–213, 1997. © 1997 Wiley-Liss, Inc.

The molecular mechanisms responsible for metastasis are not fully understood. Recently, expression of the KAI1 gene on human chromosome 11p11.2 was found to be down-regulated in metastatic prostate cancer cell lines compared with normal human prostate, suggesting that KAI1 may be a metastasis suppressor gene. The aim of this study was to investigate whether there is reduced expression of KAI1 in late-stage bladder cancer. Sixty-six paraffin-embedded bladder tissue sections were analyzed for KAI1 mRNA by in situ hybridization. Nineteen of these were from patients with no histological evidence of bladder cancer, and 47 were from papillary transitional cell carcinomas (TCCs); of these, 16 were highly invasive. KAI1 mRNA was highly expressed in the specimens of normal bladder (11 of 11; 100%), inflammatory bladder (5 of 8; 63%), and noninvasive papillary TCCs of grades 1 and 2 (15 of 24; 63%), compared to grade 3 papillary TCCs (1 of 7; 14%) or invasive TCCs (1 of 16; 6%). The differences in expression between local and invasive disease were statistically significant (P/= 0.01, chi2 test). Our results suggest that down-regulation of KAI1 mRNA is significantly associated with invasive bladder cancer and that KAI1 may represent an invasion/metastasis suppressor gene in bladder cancer.

Decreased expression of the human KAI1 metastasis-suppressor gene is involved in the progression of human prostatic cancer and possibly lung and breast cancer. To evaluate the frequency of mutation and allelic loss during the progression of human cancer, as well as to determine the regulatory mechanism for the expression of the KAI1 gene in normal and cancerous tissues, we characterized the 5'-promoter region, exon/intron organization, and transcription initiation site of the human KAI1 gene. About 80 kb of DNA was identified as the human KAI1 gene, which contains 8 kb of 5'-region, 10 exons, 9 introns, and 8 kb of DNA following exon 10. The coding region starts in exon 3 and ends in exon 10. The size of intron 1 is 29 kb, which almost equals the sizes of all other introns combined. A CpG island is present in the 5'-promoter region and extends to exon 1 and intron 1. The promoter region has no TATA or CCAAT box but has many putative binding motifs for various transcription factors, including nine Sp1 sites and five AP2 sites. These results suggest a diverse regulatory mechanism for the expression of the KAI1 gene in human tissues. The transcription initiation site of the KAI1 gene is located 181 bp upstream of the first nucleotide of the translation initiation codon. Comparisons of gene structures between KAI1 and seven other members of the transmembrane 4 superfamily revealed that the splicing sites relative to the different structural domains of the predicted proteins are well conserved, suggesting that these genes are evolutionarily related and that they arose through gene duplication and divergent evolution.