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. 1998 Aug 4;95(16):9418-23.
doi: 10.1073/pnas.95.16.9418.

In vitro loss of heterozygosity targets the PTEN/MMAC1 gene in melanoma

G P Robertson  1 , F B Furnari, M E Miele, M J Glendening, D R Welch, J W Fountain, T G Lugo, H J Huang, W K Cavenee
Affiliations

In vitro loss of heterozygosity targets the PTEN/MMAC1 gene in melanoma (V体育ios版)

G P Robertson et al. Proc Natl Acad Sci U S A. .

Abstract

Gross genetic lesions of chromosome 10 occur in 30-50% of sporadic human melanomas. To test the functional significance of this observation, we have developed an in vitro loss of heterozygosity approach in which a wild-type chromosome 10 was transferred into melanoma cells, where there was selection for its breakage and regional deletion to relieve its growth suppressive effects. The overlap of these events was at band 10q23, the site of the recently isolated PTEN/MMAC1 tumor suppressor gene, suggesting it as a potential target VSports手机版. Although the gene was expressed in the parental cells, both of its chromosomal alleles contained truncating mutations. In vitro loss of heterozygosity resulted in loss of the chromosomally introduced wild-type PTEN/MMAC1, and ectopic expression of the gene caused cell growth suppression. Thus, this approach identified PTEN/MMAC1 as a target in malignant melanoma and may provide an alternative means to localizing tumor suppressor genes. .

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Figures

Figure 1
Figure 1
Genetic analysis of microcell hybrids. (A) Partial karyotypes, derived from the four UACC 903 microcell hybrid cell lines, show the presence of three intact copies of chromosome 10 after chromosome transfer. (B) D10S1739 marker analysis for microcell hybrid Cl9(10n)2. The donor allele for D10S1739 was present in the original hybrid line and in a tumor and metastasis that derived from this hybrid. D and E indicate the locations of the donor and endogenous alleles, respectively. (C) D10S201 polymorphism analysis of UACC 903 microcell hybrids. A donor-derived D10S201 allele was present in hybrids 903(10n)24, 29, and 36 but the band intensity was reduced for hybrid 903(10n)24. (D) Fragmentation of the donor chromosome in microcell hybrid 903(10n)24. Examples of four independent partial metaphase spreads showed either an intact (Cell A), partially deleted (Cells B and C), or absent donor chromosome 10 (Cell D). Cells B and C are examples where breakage of the donor chromosome occurred at band 10q23.
Figure 2
Figure 2
Status of PTEN/MMAC1 in cell lines and microcell hybrids. (A) FISH analysis shows the presence of the chromosome 10 centromere and the PTEN/MMAC1 gene (arrow) in both copies of chromosome 10 derived from parental UACC 903 and C8161.Cl9 cell lines. ∗ indicates the signals on chromosome 9. (B) Northern blot showing PTEN/MMAC1 expression patterns in UACC 903, C8161.Cl9, human melanocytes, and MRC-5 fibroblast cells. (C) PTEN/MMAC1 sequence profiles (from nucleotides 223 to 234) derived from each cell line or microcell hybrid line. The wild-type profiles for HA(10)A and C8161.Cl9 cells are shown for comparison. ∗ shows the location of the T to G point mutation occurring in UACC 903 cells. Similar profiles are shown for each of the four UACC 903 microcell hybrids and for hybrids 903(10n)29, 36, and 37 at later passages (P number refers to cell passages). (D) Generation of an in vitro transcription and translation PTEN/MMAC1 product. A full-length 35S-labeled protein product was produced from cDNA derived from MRC-5 fibroblasts, melanocytes, HA(10)A donor cells, and all four UACC 903 microcell hybrids. The arrow shows the location of a 31-kDa species predicted to originate from methionine 134, which is associated with the next Kozak consensus sequence downstream of the truncating mutation. An 8-kDa truncated PTEN/MMAC1 product that might have arisen from the truncating mutation was not observed in UACC 903 cells. Also shown are the full-length PTEN/MMAC1 proteins produced from C8161.Cl9 cells and two microcell hybrids.
Figure 3
Figure 3
Growth suppression after transfection of PTEN/MMAC1 into human melanoma cell lines UACC 903 and C8161.Cl9. Empty vector (pBP), or vector containing wild-type PTEN/MMAC1, PTEN/MMAC1-HA, or mutant allele Y76stop-HA were transfected into (A) UACC 903 (mutant PTEN/MMAC1 background) or (B) C8161.Cl9 (wild-type PTEN/MMAC1 background), selected with puromycin for 7 days, and then counted. The number of cells remaining in the vector control was set at 100%, and all other cell numbers were normalized to this value. The results shown are combined from four separate experiments, each performed in triplicate; bars, SD. Similar results were obtained by using different plasmid preparations. (C) Ribonuclease protection analysis of RNA levels of exogenous PTEN/MMAC1 in UACC 903 and C8161.Cl9 transfected cells. The protected band at 433 bp represents an exogenous PTEN fragment that at 300 bp is a vector fragment and that at 80 bp is an 18S RNA fragment.
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