KiSS-1, a novel human malignant melanoma metastasis-suppressor gene.

BACKGROUND: Microcell-mediated transfer of chromosome 6 into human C8161 and MelJuSo melanoma cell suppresses their ability to metastasize by at least 95% without affecting their tumorigenicity. This observation demonstrates that the ability to metastasize is a phenotype distinct from tumor formation and suggests that tumorigenic cells acquire metastatic capability only after accumulating additional genetic defects. These results also imply that mutations of genes on chromosome 6 are among those late genetic changes responsible for metastatic potential. They further suggest that a melanoma metastasis-suppressor gene(s) is encoded on chromosome 6 or is regulated by genes on chromosome 6. PURPOSE: Our objective was to identify the gene(s) responsible for the suppression of metastasis in chromosome 6/melanoma cell hybrids. METHODS: A modified subtractive hybridization technique was used to compare the expression of messenger RNAs (mRNAs), via an analysis of complementary DNAs (cDNAs), in metastatic cells (C8161 or MelJuSo) and nonmetastatic hybrid clones (neo6/C8161 or neo6/MelJuSo). RESULTS: A novel cDNA, designated KiSS-1, was isolated from malignant melanoma cells that had been suppressed for metastatic potential by the introduction of human chromosome 6. Northern blot analyses comparing mRNAs from a panel of human melanoma cells revealed that KiSS-1 mRNA expression occurred only in nonmetastatic melanoma cells. Expression of this mRNA in normal heart, brain, liver, lung, and skeletal muscle was undetectable by northern blot analysis. Weak expression was found in the kidney and pancreas, but the highest expression was observed in the placenta. The KiSS-1 cDNA encodes a predominantly hydrophilic, 164 amino acid protein with a polyproline-rich domain indicative of an SH3 ligand (binds to the homology 3 domain of the oncoprotein Src) and a putative protein kinase C-alpha phosphorylation site. Transfection of a full-length KiSS-1 cDNA into C8161 melanoma cells suppressed metastasis in an expression-dependent manner. CONCLUSIONS: These data strongly suggest that KiSS-1 expression may suppress the metastatic potential of malignant melanoma cells. IMPLICATIONS: KiSS-1 may be a useful marker for distinguishing metastatic melanomas from nonmetastatic melanomas.

Tumorigenic suppression of a human cutaneous squamous cell carcinoma cell line in the nude mouse skin graft assay.

The development of human squamous cell carcinomas has been associated with a number of genetic alterations involving chromosome 11, including cytogenetic and allelic deletions as well as amplification of genes in the 11q13 region. To determine the relevance of chromosome 11 in the formation of tumors of stratified squamous epithelial origin, we have introduced, via microcell fusion, a normal human chromosome 11 into the cutaneous squamous cell carcinoma cell line A3886TGc2. The ability of chromosome 11 to modulate the tumorigenicity of A3886TGc2 was evaluated first by inoculating cells s.c. in nude mice. All hybrids remained tumorigenic but exhibited longer tumor latencies than the parent, a result previously observed by other laboratories. We then tested our epidermally derived hybrids in the more physiologically relevant environment of the nude mouse skin graft system. The tumorigenic phenotype of three of four chromosome 11 hybrids placed into nude mouse skin grafts was completely suppressed. Polymerase chain reaction amplification of DNA from normal skin present at the suppressed graft sites failed to detect the introduced human cells. This information indicates that the normal skin is of mouse origin and suggests that the chromosome 11 microcell hybrids did not differentiate in vivo, but most likely failed to survive. We propose that external environmental factors present at the site of inoculation modulate the tumorigenic potential of these cells.

Suppression of MDA-MB-435 breast carcinoma cell metastasis following the introduction of human chromosome 11.

To determine the relevance of genetic information on chromosome 11 in the development of metastatic breast tumors, we introduced a normal human chromosome 11 into the highly metastatic MDA-MB-435 breast carcinoma cell line via the microcell-mediated chromosome transfer technique. Although the MDA-MB-435 recipient cell line and four randomly selected microcell hybrid clones remained tumorigenic in nude mice, the hybrids were >95% suppressed for metastasis to lung and regional lymph nodes (p<0.01). We also tested whether chromosome 6 harbors a metastasis-suppressor gene for breast cancer as observed previously for human melanoma. Grouped together, the four neo6 microcell hybrids had no statistically significant reduction in the incidence or number of lung or lymph node metastases compared to the weakly metastatic, subcloned parent cell line, MDA-MB-453.7. Expression of nm23-H1 (NME1), a known metastasis-suppressor gene in this breast cancer cell line, did not correlate with metastasis suppression in the microcell hybrids. These results further demonstrate that control of metastasis is molecularly distinct from tumorigenic potential. They also indicate that chromosome 11 encodes a metastasis-suppressor gene for human breast cancer.

KAI1, a putative marker for metastatic potential in human breast cancer.

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, KAI1 levels were higher in chromosome 11/MDA-MB-435 cell clones. The purpose of this study was to test whether KAI1 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 KAI1 mRNA and chromosome 11 containing MDA-MB-435 (neo11/MDA-MB-435.A3 and neo11/MDA-MB-435.B1) cells had approximately twice as much KAI1 mRNA than the parental clones. MCF-10A, an immortalized normal-like non-tumorigenic mammary epithelial cell line, had the highest level of KAI1 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 KAI1 mRNA expression. These data suggest that, in addition to its role in human prostate, pancreatic and non-small cell lung cancer, KAI1 may also be a useful marker for staging human breast disease.

Free hernia surgery for the underserved is possible in the United States.

INTRODUCTION: Inguinal hernia is one of the most common ailments known to mankind. When symptomatic it can severely affect the patient's quality of life. Nevertheless, the vast majority of inguinal herniorrhaphies are elective and, therefore, not available to uninsured patients who do not have the financial wherewithal to pay for the operation. Using the Surgery on Sunday model developed in Kentucky, hernia repair for the underserved developed a free clinic for hernia surgery, based on institutional commitment to the poor as well as the volunteer efforts of medical students and hospital personnel at all levels. METHODS: After consulting with Surgery on Sunday personnel, HRFU determined the number of in need patients by consulting with local free clinic physicians. Second, and most time consuming, was the application for the Federal Tort Claims Act (FTCA) medical legal protection. Under this law, all in hospital credentialed volunteer professionals are medico-legally protected if the surgery is performed in an associated free clinic. After FTCA application re-writes and committee meetings to work out logistics of the pre-op clinic, the follow-up clinic, enlistment of other volunteers such as transporters, translators, housekeeping for the ORs, a pharmacist, registration personnel and creation of HRFU hospital forms we established a surgery date. A memorandum of understanding was drafted and an agreement letter with the hospital system was co-signed. Fourteen patients were seen in the pre-operative clinic and two were placed on waiting list. Patients were operated upon using 3 operating rooms and a volunteer staff of 4 surgeons, 4 anesthesia personnel and 13 nurses. RESULTS: No surgical complications were encountered intra-operatively or in the recovery room, and all patients were discharged by 2:30 p.m. 1 week post-operatively one patient had severe incisional pain, two had operative site swelling, but there was no evidence of infection or hematoma, and one had a distal sac fluid collection. All patients returned for follow-up and were appreciative of the care provided. The enthusiasm and participation of the patients and staff both pre-operatively, the day of surgery and postoperatively was outstanding. CONCLUSION: On the basis of this result HRFU is prepared to assist other US hernia specialists and their respective hospitals to make Surgery on Sunday a possibility in their community.

Correlation between reduction of metastasis in the MDA-MB-435 model system and increased expression of the Kai-1 protein.

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.

Localization of a tumor suppressor gene in 11p15.5 using the G401 Wilms' tumor assay.

Multiple studies have underscored the importance of loss of tumor suppressor genes in the development of human cancer. To identify these genes, we used somatic cell hybrids in a functional assay for tumor suppression in vivo. A tumor suppressor gene in 11p15.5 was detected by transferring single human chromosomes into the G401 Wilms' tumor cell line. In order to better map this gene, we created a series of radiation-reduced t(X;11) chromosomes and characterized them at 24 loci between H-RAS and beta-globin. Interestingly, three of the chromosomes were indistinguishable as determined by genomic and cytogenetic analyses. Each contains an interstitial deletion with one breakpoint in 11p14.1 and the other breakpoint between the D11S601 and D11S648 loci in 11p15.5. PFGE analysis localized the 11p15.5 breakpoints to a 175 kb MluI fragment that hybridized to D11S601 and D11S648 probes. Genomic fragments from this 175 kb region were hybridized to DNA from mouse hybrid lines containing the delta t(X;11) chromosomes. This analysis detected the identical 11p15.5 breakpoint which disrupts a 7.8 kb EcoRI fragment in all three of the delta t(X;11) chromosomes, suggesting they are subclones of the same parent colony. Upon transfer into G401 cells, one of the chromosomes suppressed tumor formation in nude mice, while the other two chromosomes lacked this ability. Thus, our mapping data indicate that the gene in 11p15.5 which suppresses tumor formation in G401 cells must lie telomeric to the D11S601 locus. Koi et al. (Science 260: 361-364, 1993) have used a similar functional assay to localize a growth suppressor gene for the RD cell line centromeric to the D11S724 locus. The combination of functional studies by our lab and theirs significantly narrows the location of the tumor suppressor gene in 11p15.5 to the approximately 500 kb region between D11S601 and D11S724.