Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma.

Deciphering the molecular basis of cancer is critical for developing novel diagnostic and therapeutic strategies. To better understand the early molecular events involving osteogenic sarcoma (OGS), we have initiated a program to identify potential tumor suppressor genes. Expression profiling of total RNA from ten normal bone cell lines and eleven OGS-derived cell lines by microarray showed 135-fold lower expression of FRZB/sFRP3 mRNA in OGS cells compared to bone cells; this down-regulation of Frzb/sFRP3 mRNA expression was found to be serum-independent. Subsequently, fourteen OGS biopsy specimens showed nine-fold down-regulation of Frzb/sFRP3 mRNA expression compared to expression in eight normal bone specimens as determined by microarray. FRZB /sFRP3 protein level was also found to be at a very low level in 4/4 OGS cell lines examined. Quantitation by RT-PCR indicated approximately 70% and approximately 90% loss of Frzb/sFRP3 mRNA expression in OGS biopsy specimens and OGS-derived cell lines respectively, compared to expression in bone (p<0.0001). Hybridization experiments of a cDNA microarray containing paired normal and tumor specimens from nineteen different organs did not show any significant difference in the level of Frzb/sFRP3 mRNA expression between the normal and the corresponding tumor tissues. Exogenous expression of FRZB/sFRP3 mRNA in two OGS-derived cell lines lacking endogenous expression of the mRNA produced abundant mRNA from the exogenous gene, eliminating degradation as a possibility for very low level of FRZB/sFRP3 mRNA in OGS specimens. Results from PCR-based experiments suggest that the FRZB/sFRP3 gene is not deleted in OGS cell lines, however, karyotyping shows gross abnormalities involving chromosome 2 (location of the FRZB gene) in five of twelve OGS-derived cell lines. Together, these data suggest a tumor-suppressive potential for FRZB/sFRP3 in OGS.

The human homolog of yeast SEP1 is a novel candidate tumor suppressor gene in osteogenic sarcoma.

The hSEP1 gene is the human homolog of yeast SEP1. Yeast SEP1 is a multifunctional gene that regulates a variety of nuclear and cytoplasmic functions including homologous recombination, meiosis, telomere maintenance, RNA metabolism and microtubule assembly. The function of hSEP1 is not known. We show loss or reduced expression of hSEP1 messenger RNA (mRNA) in three of four primary osteogenic sarcoma (OGS)-derived cell lines and in eight of nine OGS biopsy specimen. In addition, we find a heterozygous missense mutation (Valine(1484)>Alanine) at a conserved amino acid in the primary OGS-derived cell line U2OS. Importantly, we identified a homozygous missense mutation involving a CG-dinucleotide leading to a change in a conserved amino acid, aspartic acid(1137) >asparagine, in the primary OGS-derived cell line, TE85. hSEP1 mRNA expression was nearly undetectable in TE85 and low in U2OS cell lines. None of these mutations were identified in 20 normal samples consisting of bone, cartilage and fibroblast. The hSEP1 gene is located in chromosome 3 at 3q25-26.1 between markers D3S1309 and D3S1569. An adjacent locus defined by the polymorphic markers D3S1212 and D3S1245 has previously been reported to undergo loss of heterozygosity (LOH) at a >70% frequency in OGS and claimed to harbor an important tumor suppressor gene in osteosarcoma. The homozygous mutation in the hSEP1 mRNA in TE85 cell line suggest that this gene itself is subject to LOH. Taken together, these results suggest that hSEP1 acts as a tumor suppressor gene in OGS.