Fibulin-5 protects the extracellular matrix of chondrocytes by inhibiting the Wnt/ß-catenin signaling pathway and relieves osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a common disease in the elderly and seriously affects the quality of life of patients. The purpose of this study was to explore the protective effect of Fibulin-5 on articular chondrocytes and its mechanism of action. PATIENTS AND METHODS: Articular cartilage tissues from patients with OA and normal people were selected and tested for differences in Fibulin-5 expression. In addition, human chondrocytes were cultured, and the effects of Fibulin-5 on the extracellular matrix (ECM) of chondrocytes and the level of inflammation were examined by means of cell transfection and cytokine intervention. SKL2001, an agonist of the Wnt/ß-catenin signaling pathway, was used to validate the mechanism of action of Fibulin-5 to protect chondrocytes. RESULTS: Fibulin-5 was lowly expressed in the cartilage tissue of patients with OA. Overexpression of Fibulin-5 significantly increased the expressions of ECM collagen II and aggrecan in chondrocytes, while decreasing the expressions of MMP-3 and MMP-13. In addition, Fibulin-5 reduced IL-1ß-induced inflammation of chondrocytes, as well as expressions of IL-6, IL-8, and TNF-α. Overexpression of Fibulin-5 also reduced the activity of Wnt/ß-catenin signaling pathway, and activation of Wnt/ß-catenin signaling pathway attenuated the protective effects of Fibulin-5 on the ECM of chondrocytes. CONCLUSIONS: Fibulin-5 can protect the ECM of chondrocytes and reduce the inflammatory response of chondrocytes by inhibiting the Wnt/ß-catenin signaling pathway.

[Clinical study of a new wearable device for rehabilitation after total knee arthroplasty].

Objective: To evaluate the effect of promoting knee joint rehabilitation after total knee arthroplasty (TKA) with a rehabilitation training instrument NEO-GAIT. Methods: Sixty patients who received TKA from January 2017 to July 2017 in the Third Hospital of Hebei Medical University were randomly assigned to receive rehabilitation training with continuous passive motion (CPM) or NEO-GAIT with random number (30 cases in CPM group, included 8 males and 22 females; 30 cases in NEO-GAIT group, included 6 males and 24 females). The visual analogue scale (VAS) evaluation of pain, the postoperative range of motion (ROM) of the knee at the 5th and 10th day, and the Hospital for Special Surgery Knee-rating Score (HSS) at 1-month and 3-month follow-up were recorded. The data were compared between the two groups with paired t test. Results: All the patients were followed-up for more than 3 months. The mean VAS in CPM group and NEO-GAIT group on the 5th day was 2.4±1.1, 2.8±1.3, respectively; and it was 2.1±1.1, 2.5±1.2 respectively on the 10th day after the operation (t=-1.618, -1.505, both P>0.05). There was no significant difference in ROM on the 5th day after operation between the 2 groups (84°±12° vs 85°±12°, t=-0.377, P>0.05); however, it was remarkably higher in the NEO-GAIT group (95°±11°) than that in CPM group (88°±8°) on the 10th day after the operation (t=-3.002, P<0.05). The HSS score at 1-month follow-up in CPM group was 72±9, and it was 84±10 in NEO-GAIT group (t=-5.358, P<0.05); but it was comparative between the two groups at the 3-month follow-up (87±5 vs 89±5, t=-1.575, P>0.05). Conclusion: NEO-GAIT plays a more active and effective role in promoting postoperative rehabilitation after TKA than CPM.

The study on specific umbilical blood Dc vaccine for Beige nude mice loaded human colorectal carcinoma to induce anti-tumor immunity.

OBJECTIVE: This study is to observe the immunosuppression of CD137L transfected umbilical blood Dcs (Dendritic cell) vaccine to tumor development of SCID/ Beige nude mice. MATERIALS AND METHODS: Samples of umbilical blood in the childbirth pregnant women were collected by density gradient centrifugation. Umbilical cord blood dendritic cells (Dcs) were transfected by specific CD137L via LipofectamineTM method and cells were harvested. Meanwhile, the peripheral blood of volunteers was collected to isolate Dcs, the Dcs were cultured for 5 days and hatched with SW-1116 cells antigen. The mature Dcs were harvested. The male SCID/Beige nude mice were subcutaneously injected with human SW-1116 cells in axillary to build colorectal carcinoma model as blank control (Blank). The naked peripheral blood Dc vaccine group (cPBMCs), the SW-1116 antigen-specific peripheral blood Dc vaccine group (pDcs) and the CD137L specific umbilical blood Dc vaccine group (tuDcs) were injected 24 h before tumor cells injection, respectively to recur the humanized immune reconstruction. The general life, living habits changes, tumor growing time and tumor size were observed. The nude mice were sacrificed 18 days after tumor formation. The tumor size, mice weight, in vitro tumor weight, liver weight and spleen weight of mice were recorded to evaluate the anti-tumor effect of the specific immune cells. RESULTS: The nude mice in pDcs group showed better general living condition, slower tumor growth, smaller tumor volume and no ulceration, necrosis, and death in nude mice. The tumor formation time in different groups was 4.71 ± 0.18 ds (blank), 7.71 ± 0.29 ds (cPBMCs), 7.86 ± 0.26 ds (pDcs) and 8.14 ± 0.69 ds (tuDcs) respectively. There were significant differences between blank and other three groups (F = 40.96, p < 0.01). Compared to mice in blank group, the tumor volume of cPBMCs group was significantly smaller (201.43 ± 69.84 mm³ vs. 436.04 ± 54.50 mm³, p < 0.01) and the tumor weight were significantly smaller (1.25 ± 0.12 g vs. 2.83 ± 0.24 g, p < 0.01). The tumor volume of tuDcs mice was significantly smaller than that of blank (92.11 ± 11.55 mm³ vs. 436.04 ± 54.50 mm³, p < 0.01) and cPBMCs mice (92.11 ± 11.55 mm³ vs. 201.43 ± 69.84 mm³, p < 0.01). Similarly, the tumor weight of tuDcs mice was significantly smaller than that of blank (0.66 ± 0.07 g vs. 2.83 ± 0.24 g, p < 0.01) and cPBMCs mice (0.66 ± 0.07 g vs. 1.25 ± 0.12 g, p < 0.01). There was no significant difference in tumor volume (92.11 ± 11.55 mm³ vs. 85.61 ± 11.59 mm³, p = 0.69) and tumor weight (0.66 ± 0.07 g vs. 0.63 ± 0.09 g, p = 0.75) between tuDcs group and pDcs group. CONCLUSIONS: The specific CD137L transfected umbilical blood Dc vaccine had significant anti-tumor effect against human colon cancer in nude mice via increasing the number of immune effector cell in tumor microenvironment.

Krüppel-like factor 5 promotes breast cell proliferation partially through upregulating the transcription of fibroblast growth factor binding protein 1.

The Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor promoting cell proliferation, cell-cycle progression and survival. A high expression level of KLF5 mRNA has been shown to be associated with shorter breast cancer patient survival. However, the mechanism of KLF5 action in breast cancer is still not clear. In this study, we found that both KLF5 and its downstream gene fibroblast growth factor binding protein 1 (FGF-BP) are co-expressed in breast cell lines and primary tumors. Manipulation of the KLF5 expression can positively regulate the FGF-BP mRNA and protein levels in multiple breast cell lines. In addition, the secreted FGF-BP protein in the conditional medium is also regulated by KLF5. Furthermore, we demonstrated that KLF5 binds and activates the FGF-BP promoter through a GC box by luciferase reporter, oligo pull down and chromatin immunoprecipitation (ChIP) assays. When FGF-BP is depleted by siRNA, KLF5 fails to promote cell proliferation in MCF10A, SW527 and TSU-Pr1. We further demonstrated that overexpression or addition of FGF-BP rescues the KLF5-knockdown-induced growth arrest in MCF10A cells. Finally, KLF5 significantly promotes MCF7 breast cancer cell xenograft growth in athymic nude mice. These findings suggest that KLF5 may promote breast cancer cell proliferation at least partially through directly activating the FGF-BP mRNA transcription. Understanding the mechanism of KLF5 action in breast cancer may result in useful diagnostic and therapeutic targets.

Ubiquitin E3 ligase WWP1 as an oncogenic factor in human prostate cancer.

The gene for E3 ubiquitin ligase WWP1 is located at 8q21, a region frequently amplified in human cancers, including prostate cancer. Recent studies have shown that WWP1 negatively regulates the TGFbeta tumor suppressor pathway by inactivating its molecular components, including Smad2, Smad4 and TbetaR1. These findings suggest an oncogenic role of WWP1 in carcinogenesis, but direct supporting evidence has been lacking. In this study, we examined WWP1 for gene dosage, mRNA expression, mutation and functions in a number of human prostate cancer samples. We found that the WWP1 gene had copy number gain in 15 of 34 (44%) xenografts and cell lines from prostate cancer and 15 of 49 (31%) clinical prostate cancer samples. Consistently, WWP1 was overexpressed in 60% of xenografts and cell lines from prostate cancer. Mutation of WWP1 occurred infrequently in prostate cancer. Functionally, WWP1 overexpression promoted colony formation in the 22Rv1 prostate cancer cell line. In PC-3 prostate cancer cells, WWP1 knockdown significantly suppressed cell proliferation and enhanced TGFbeta-mediated growth inhibition. These findings suggest that WWP1 is an oncogene that undergoes genomic amplification at 8q21 in human prostate cancer, and WWP1 overexpression is a common mechanism involved in the inactivation of TGFbeta function in human cancer.

Loss of heterozygosity at 13q14 and 13q21 in high grade, high stage prostate cancer.

BACKGROUND: Loss of heterozygosity (LOH) at chromosome 13q has been frequently detected in prostate cancer, and three regions (i.e., 13q14, 13q21, and 13q33) may harbor tumor suppressor genes important in this neoplasm. In this study, we examined the frequency of 13q LOH in advanced prostate cancers, in order to determine the clinicopathologic relevance of 13q LOH. METHODS: LOH was determined by analyzing microsatellite markers in 41 cases of microdissected predominantly high grade prostate cancer tissues and their matched nonneoplastic cells. The results were compared with those generated previously for lower grade, asymptomatic cancers. RESULTS: The frequencies of LOH at 13q14, 13q21, and 13q33 were 62% (21/34), 57% (20/35), and 34% (11/32), respectively. In comparison to previous results, LOH at 13q14 and 13q21 but not 13q33 was more frequent in prostate cancers that produced local clinical symptoms (bladder outlet obstruction) than those that did not (P < 0.05). LOH at 13q14 was also significantly more frequent in high grade and high stage cancers than those that were lower grade and lower stage (P < 0.05). CONCLUSIONS: Although the target genes on 13q have not been identified in carcinomas of the prostate, LOH at 13q14 in particular is associated with clinically significant prostate cancers. Further fine mapping of these loci may lead to identification of tumor suppressor genes that are deleted in aggressive carcinomas of the prostate.

KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma.

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.

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas.

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.

Defining a common region of deletion at 13q21 in human cancers.

Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus. In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162. At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification. To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers. The contig consisted of 75 overlapping BAC clones. In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average). The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region. A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation. In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing. These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer. The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21.

Mutations of PTEN/MMAC1 in primary prostate cancers from Chinese patients.

PTEN/MMAC1 is a putative tumor suppressor gene located on 10q23, one of the most frequently deleted chromosomal regions in human prostate cancer. Although mutations of PTEN have often been detected in metastases of prostate cancer, localized tumors have shown lower rates of mutation, which have varied from 0 to 20% among different studies. It is unknown whether the rate of PTEN mutations is different in prostate cancer from Asian men compared with Western men. To further clarify the role of PTEN in prostate cancer and to examine the gene for mutations in Asian men, we analyzed 32 cases of primary prostate cancers from Chinese patients, each of whom was not diagnosed by screening with serum prostate-specific antigen, for PTEN mutations using the methods of tissue microdissection, single-strand conformational polymorphism, and direct DNA sequencing. Seventy % of the tumors were Gleason scores 8-10, whereas the remainder were Gleason score 7. Six metastases of prostate cancer from American patients were also analyzed. Five of 32 (16%) primary prostate cancers from Chinese men and two of six metastases from American men showed mutations in a total of 10 codons of PTEN, which involved exons 1, 2, 5, 8, and 9. Two of the mutations were truncation type, whereas the rest were missense mutations. The mutation frequency in these cases from Asian patients was higher than that in our previous study of cases in radical prostatectomy specimens from American men, in which the 40 primary tumors were lower grade and had been detected by serum prostate-specific antigen test. We conclude that mutation of PTEN occurs more often in primary prostate cancers of Chinese men, whose tumors are high grade and reflective of an unscreened population.

Chromosomal deletions and tumor suppressor genes in prostate cancer.

Chromosomal deletion appears to be the earliest as well as the most frequent somatic genetic alteration during carcinogenesis. It inactivates a tumor suppressor gene in three ways, that is, revealing a gene mutation through loss of heterozygosity as proposed in the two-hit theory, inducing haploinsufficiency through quantitative hemizygous deletion and associated loss of expression, and truncating a genome by homozygous deletion. Whereas the two-hit theory has guided the isolation of many tumor suppressor genes, the haploinsufficiency hypothesis seems to be also useful in identifying target genes of chromosomal deletions, especially for the deletions detected by comparative genomic hybridization (CGH). At present, a number of chromosomal regions have been identified for their frequent deletions in prostate cancer, including 2q13-q33, 5q14-q23, 6q16-q22, 7q22-q32, 8p21-p22, 9p21-p22, 10q23-q24, 12p12-13, 13q14-q21, 16q22-24, and 18q21-q24. Strong candidate genes have been identified for some of these regions, including NKX3.1 from 8p21, PTEN from 10q23, p27/Kip1 from 12p13, and KLF5 from 13q21. In addition to their location in a region with frequent deletion, there are functional and/or genetic evidence supporting the candidacy of these genes. Thus far PTEN is the most frequently mutated gene in prostate cancer, and KLF5 showed the most frequent hemizygous deletion and loss of expression. A tumor suppressor role has been demonstrated for NKX3.1, PTEN, and p27/Kip1 in knockout mice models. Such genes are important targets of investigation for the development of biomarkers and therapeutic regimens.

Deletion at 13q21 is associated with aggressive prostate cancers.

Previous cytogenetic and molecular genetic analyses suggest that the q21 band of chromosome 13 harbors a tumor suppressor gene(s) involved in prostatic carcinogenesis. The precise genetic location, however, has not been defined. In this study, we examined prostate cancer specimens and cell lines/xenograft for genetic deletions at 13q21, using the methods of tissue microdissection and duplex PCR. Deletions at 13q21 were detected in 13 of 147 (9%) prostate cancer samples. Deletion of the same region was also detected in the LNCaP cell line and the PC-82 xenograft of prostate cancer. The overlapping region of deletion in LNCaP and PC-82 spans 3.1 cM or 2.9 cR, which is equivalent to 1-3 Mb. The endothelin receptor B gene, a possible tumor suppressor gene at 13q21, was not located in the region of deletion. Among the 13 prostate neoplasms with deletion at 13q21, 5 were metastases, and 7 were poorly differentiated primary tumors. The only primary tumor that was not poorly differentiated but had deletion occurred in one of the youngest patients (49 years) at diagnosis. These results provide evidence that 13q21 may harbor an unidentified gene(s) whose inactivation occurs in some aggressive carcinomas of the prostate. In addition, this study provides a framework for the cloning and identification of the 13q21 gene(s).

Frequent down-regulation and lack of mutation of the KAI1 metastasis suppressor gene in epithelial ovarian carcinoma.

OBJECTIVE: KAI1 is a recently identified metastasis suppressor gene on human chromosome 11p11.2. It belongs to a structurally distinct family of cell surface glycoproteins. Decreased KAI1 expression seems to be involved in the progression of human prostate, lung, pancreatic, and possibly breast cancer, and recently a reduced KAI1 protein expression has been demonstrated in several ovarian carcinoma cell lines. The aim of this study is to determine whether the KAI1 gene is altered in human epithelial ovarian carcinomas. In addition, its prognostic significance in this tumor is also evaluated. METHODS: To detect KAI1 expression, 102 tumor samples from benign, borderline, primary invasive, metastatic, and recurrent epithelial ovarian tumors were prepared for immunohistochemical study with C-16, an anti-KAI1 polyclonal antibody. In addition, cellular RNA from 24 primary invasive and 7 recurrent tumors was also analyzed for KAI1 expression by using a reverse transcriptase PCR (RT-PCR) technique. The PCR single-strand conformation polymorphism method and direct DNA sequencing were used to detect KAI1 mutation in the 44 primary invasive and 8 recurrent ovarian carcinomas. RESULTS: In immunohistochemical study, decrease of KAI1 protein expression was associated with the progression of ovarian tumor. However, it had no relation to the stage of primary invasive cancers because of its frequent occurrence in early stage tumors. KAI1 expression was also frequently down-regulated in primary invasive and recurrent tumors in RT-PCR analysis. Except for a missense change at codon 241 (ATC to GTC), which causes the substitution of a valine for an isoleucine in the amino acid sequence and occurs in both normal and tumor tissues, no mutation of the KAI1 gene was found in any of the 52 carcinomas. Although there was a trend for deteriorating survival from patients with KAI1-preserved tumors to those with KAI1-decreased and -negative tumors, statistically it was not significant (P = 0.079). CONCLUSION: KAI1 may play a role in the malignant progression of epithelial ovarian carcinoma through the down-regulation of expression rather than gene mutation. Since the decreased expression presented frequently in early stage tumors, it may be an early event in the progression of this tumor and its prognostic significance needs further investigation with a larger number of cases.

Loss of heterozygosity and lack of mutations of the XPG/ERCC5 DNA repair gene at 13q33 in prostate cancer.

BACKGROUND: Three regions of chromosome 13 were previously identified for having loss of heterozygosity (LOH) in human prostate cancer. One of them, at 13q33, was defined by LOH at markers D13S158 and D13S280. The XPG/ERCC5 gene, a DNA repair gene that when mutated in the germline leads to xeroderma pigmentosum, has been mapped to 13q33, within one megabase of D13S158 and D13S280. This paper describes LOH and mutational analysis of the XPG gene in human prostate cancers, in order to determine whether the XPG gene is involved in the development of prostate cancer. METHODS: LOH of the XPG gene was analyzed in 40 primary prostate cancers and 14 metastases by using the microsatellite assay, and its mutations were examined in 5 cell lines, 14 metastases, and 8 tumors with LOH at 13q33 by using the single-strand conformation polymorphism (SSCP)-direct DNA sequencing analysis. RESULTS: Four of the 29 (14%) informative primary tumors and 4 of 8 (50%) metastases showed LOH for the XPG gene. Analysis of the 8 tumors with LOH at the 13q33 region, 14 metastases, and 5 cell lines of prostate cancer revealed two polymorphisms but no mutation of the gene. The polymorphism in exon 2 did not change the amino-acid sequence of the XPG protein, but the exon 15 polymorphism altered codon 1104 from histidine to aspartic acid. The two polymorphisms also occurred in individuals without prostate cancer. CONCLUSIONS: LOH at XPG in prostate cancer supports the conclusion that the 13q33 region contains a gene important in the development of prostate cancer, while lack of mutations of the gene suggests that XPG is not the target gene involved.

Three distinct regions of allelic loss at 13q14, 13q21-22, and 13q33 in prostate cancer.

Chromosome 13 is one of the most frequently altered chromosomes in cancer, including carcinoma of the prostate. Two known tumor suppressor genes, RB1 and BRCA2, map to chromosome 13; however, recent reports suggest that unknown genes on 13q are more likely to be involved in the development of prostate cancer. In order more fully to define the genetic changes on chromosome 13 in prostate neoplasms, we analyzed 27 polymorphic microsatellite markers spanning the q arm for loss of heterozygosity in 40 primary tumors and in metastases from 11 other patients who died of prostate cancer. Of the 40 primary tumors, 23 (58%) showed LOH for at least one marker. Three distinct regions at q14, q21-22, and q33, defined by markers D13S267-->D13S153, D13S166-->D13S1225, and D13S259-->D13S274, showed the most frequent LOH, suggesting their involvement in the development of prostate cancer. For the 12 patients whose tumors showed LOH at these markers, the average age at diagnosis was 58 years, which was younger than that (63 years, P < 0.05) for the 28 patients whose tumors lacked LOH. Ten of the 11 (91%) metastases showed LOH with one or more markers. Two of the three most frequently deleted regions (i.e., q14 and q21-22) in the primary tumors and markers linked to the RB1, BRCA2, and EDNRB genes showed high frequencies (56-71%) of LOH in metastases. These results demonstrate that allelic loss on chromosome 13 at q14, q21-22, and q33 occurs in a subset of primary prostate tumors and is a frequent event in metastatic lesions of prostate cancer.

Methylation of the CD44 metastasis suppressor gene in human prostate cancer.

Previous studies demonstrated that CD44 is a metastasis suppressor gene for prostate cancer and that the expression of CD44 both at mRNA and protein levels is down-regulated during prostate cancer progression, with down-regulation being correlated with higher tumor grade, aneuploidy, and distant metastasis. In this study, we evaluated DNA hypermethylation as a potential mechanism accompanying this decreased CD44 expression in human prostate cancer. Nucleotide sequence analysis revealed a CpG island in the CD44 transcriptional regulatory region. We found that cytosine methylation of CD44 promoter occurs in CD44-negative prostate cancer cell line (i.e., LNCaP) but not in prostate cancer cell lines (i.e., TSU, PC3, and DU145) expressing this gene. In addition, we examined methylation status of CD44 in 84 matched normal and cancer prostate specimens. Hypermethylation of the 5' CpG island of CD44 gene was observed in 31 of 40 primary prostate cancer specimens, 3 of 4 distant organ site metastases obtained at autopsy from men who died of prostate cancer, and 4 of the 40 matched normal tissues. These results demonstrated that methylation of the 5' CpG island of CD44 gene is closely associated with transcriptional inactivation, resulting in a decreased expression of CD44 in human prostate cancer.

Identification of the rat homologue of KAI1 and its expression in Dunning rat prostate cancers.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

PTEN/MMAC1 is infrequently mutated in pT2 and pT3 carcinomas of the prostate.

Deletion of the q23-24 region of human chromosome 10 is one of the most frequent genetic alterations in prostate cancer, suggesting that inactivation of a tumor suppressor gene in this region is involved in the development or progression of this carcinoma. A candidate gene, PTEN/MMAC1, has been identified from this chromosomal region; mutations of this gene have been found in various advanced tumors and cell lines including those of prostate cancer. To further define the role of PTEN/MMAC1 in the development of prostate cancer and its spectrum of genetic alterations, we analysed 40 pT2 or pT3 prostate tumors for allelic loss, mutations, and homozygous deletions using PCR-based methods. Six tumors showed loss of heterozygosity for one of the ten markers analysed, while one tumor showed loss of two markers. None of the markers within PTEN/MMAC1 was lost. Direct sequencing of PCR amplified exons and intron/exon junctions of all 40 tumors revealed three sequence variants, one of which was a point mutation in exon 9, while the other two were polymorphisms. Using multiplex PCR, no homozygous deletions were detected in any of the neoplasms. Our results showing a low frequency of alterations of PTEN/MMAC1 in pT2 and pT3 prostate cancers suggest that this gene plays an insignificant role in the development of most low stage carcinomas of the prostate.

Location of KAI1 on the short arm of human chromosome 11 and frequency of allelic loss in advanced human prostate cancer.

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.

Genomic organization of the human KAI1 metastasis-suppressor gene.

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.