Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression - PubMed (original) (raw)

Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression

Y E Whang et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. We examined PTEN/MMAC1 in advanced prostate cancer for gene mutations or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1-specific antibody. Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and reverse transcription-PCR of mRNA. PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by in vitro treatment with the demethylating agent 5-azadeoxycytidine. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblot analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. These results demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer.

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Figures

Figure 1

Figure 1

Homozygous deletion of PTEN/MMAC1. (A) Map of homozygous deletions. The approximate location of the PTEN/MMAC1 gene is shown as an open box. The ordered set of microsatellite markers on chromosome 10q23 are shown above the line. +, the presence of at least one allele as indicated by amplification of the correct size fragment by PCR of genomic DNA; −, the deletion of both alleles as indicated by the absence of the amplified fragment after PCR of genomic DNA. (B) Southern blot analysis. The filter containing _Hin_dIII-digested genomic DNA separated by electrophoresis was probed with the PTEN exon 5 probe. The band representing the pseudogene is indicated by ψ. The band representing the functional PTEN/MMAC1 gene is indicated by PTEN.

Figure 2

Figure 2

Northern blot analysis of PTEN/MMAC1 expression. (A) Total RNA from indicated cell lines and xenografts was analyzed by Northern blot analysis by using a 1.2-kb PTEN/MMAC1 probe. LAPC-4 AD and LAPC-4 AI represent the androgen-dependent and androgen-independent sublines, respectively. (B) The same Northern filter was stripped and reprobed for expression of glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Figure 3

RT-PCR analysis of PTEN/MMAC1 expression. (A) Expression of PTEN/MMAC1, PSA, and human β-actin in indicated cell lines and xenografts was analyzed by RT-PCR, and ethidium bromide-stained agarose gels are shown. Because the PCR primers for β-actin are directed against the human sequence, the signal in murine NIH 3T3 cells is less pronounced than in other lanes. (B) Expression of PTEN/MMAC1 in peripheral blood leukocytes (normal 12) and PTEN/MMAC1 and PSA in primary prostate tumor tissues before establishment of xenografts was analyzed by RT-PCR. Tumors 12, 14, and 15 were used to establish LAPC-12, 14, and 15, respectively.

Figure 4

Figure 4

Restoration of PTEN/MMAC1 expression by 5-azadeoxycytidine. LuCaP-35 xenograft cells cultured in vitro were treated with 5-azadeoxycytidine at a concentration of 0, 1, or 2 μM, and cells were harvested for RNA after 4 days. Expression of PTEN/MMAC1 was detected by RT-PCR and was visualized by ethidium bromide staining. RT-PCR of PSA is shown as a control for the integrity of RNA in all samples.

Figure 5

Figure 5

PTEN/MMAC1 protein expression. (A) Immunoblot of PTEN/MMAC1 protein. Rabbit polyclonal antibody raised against the C terminus of PTEN/MMAC1 protein was used to probe the whole cell extracts. Rat1-PTEN cells were infected with recombinant retrovirus expressing a cDNA for the PTEN/MMAC1 coding region. Cell extracts from normal prostate tissues from three different individuals (denoted as 1, 2, 3) are shown along with cell extracts from xenografts (LAPC-3, -4, -9, -14). The full length PTEN/MMAC1 protein is indicated by a solid arrow. (B) Immunohistochemical localization of PTEN/MMAC1 in LAPC-4 xenograft tumor (Right) reveals cytoplasmic staining with immunoperoxidase technique (brown). No staining is seen in LAPC-9 (Left) run in parallel on the same slide.

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