Identification of a novel serine protease-like gene, the expression of which is down-regulated during breast cancer progression

Xiao Long Liu, David E. Wazer, Kiyoko Watanabe, Vimla Band

Research output: Contribution to journalArticle

155 Citations (Scopus)

Abstract

In an effort to isolate genes with down-regulated expression at the mRNA level during oncogenic transformation of human mammary epithelial cells (MECs), we performed subtractive hybridization between normal MEC strain 76N and its radiation-transformed tumorigenic derivative 76R-30. Here, we report the isolation of cDNA clones corresponding to a 1.4-kb mRNA species that is abundantly expressed in 76N cells but is drastically reduced in 76R-30 cells. Based on its selective expression in MECs compared with fibroblasts, the corresponding gene is designated NES1 (normal epithelial cell-specific 1). Sequence analysis of the full-length NES1 cDNA clones revealed it to be a novel gene with a predicted polypeptide of 30.14 kilodaltons; in vitro transcription and translation confirmed this prediction. Database searches revealed a 50-63% similarity and 34-42% identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor γ, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator). Importantly, all of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, suggesting that it may be a protease. An antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detected a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive MECs, suggesting that NESt is a secreted protein. The 1.4-kb NES1 mRNA was expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovary; a 1.1-kb transcript was found in the pancreas. Although expression of the NES1 mRNA was observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines showed a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorigenesis.

Original languageEnglish (US)
Pages (from-to)3371-3379
Number of pages9
JournalCancer Research
Volume56
Issue number14
StatePublished - Jul 15 1996

Fingerprint

Serine Proteases
Epithelial Cells
Breast Neoplasms
Gene Expression
Breast
Messenger RNA
Peptide Hydrolases
Peptides
Genes
Pancreas
Ovary
Carcinogenesis
Proteins
Complementary DNA
Clone Cells
Kringles
Tissue Kallikreins
Nerve Growth Factor
Tissue Plasminogen Activator
Prostate-Specific Antigen

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Identification of a novel serine protease-like gene, the expression of which is down-regulated during breast cancer progression. / Liu, Xiao Long; Wazer, David E.; Watanabe, Kiyoko; Band, Vimla.

In: Cancer Research, Vol. 56, No. 14, 15.07.1996, p. 3371-3379.

Research output: Contribution to journalArticle

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abstract = "In an effort to isolate genes with down-regulated expression at the mRNA level during oncogenic transformation of human mammary epithelial cells (MECs), we performed subtractive hybridization between normal MEC strain 76N and its radiation-transformed tumorigenic derivative 76R-30. Here, we report the isolation of cDNA clones corresponding to a 1.4-kb mRNA species that is abundantly expressed in 76N cells but is drastically reduced in 76R-30 cells. Based on its selective expression in MECs compared with fibroblasts, the corresponding gene is designated NES1 (normal epithelial cell-specific 1). Sequence analysis of the full-length NES1 cDNA clones revealed it to be a novel gene with a predicted polypeptide of 30.14 kilodaltons; in vitro transcription and translation confirmed this prediction. Database searches revealed a 50-63{\%} similarity and 34-42{\%} identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor γ, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator). Importantly, all of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, suggesting that it may be a protease. An antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detected a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive MECs, suggesting that NESt is a secreted protein. The 1.4-kb NES1 mRNA was expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovary; a 1.1-kb transcript was found in the pancreas. Although expression of the NES1 mRNA was observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines showed a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorigenesis.",
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N2 - In an effort to isolate genes with down-regulated expression at the mRNA level during oncogenic transformation of human mammary epithelial cells (MECs), we performed subtractive hybridization between normal MEC strain 76N and its radiation-transformed tumorigenic derivative 76R-30. Here, we report the isolation of cDNA clones corresponding to a 1.4-kb mRNA species that is abundantly expressed in 76N cells but is drastically reduced in 76R-30 cells. Based on its selective expression in MECs compared with fibroblasts, the corresponding gene is designated NES1 (normal epithelial cell-specific 1). Sequence analysis of the full-length NES1 cDNA clones revealed it to be a novel gene with a predicted polypeptide of 30.14 kilodaltons; in vitro transcription and translation confirmed this prediction. Database searches revealed a 50-63% similarity and 34-42% identity with several families of serine proteases, in particular the trypsin-like proteases, members of the glandular kallikrein family (including prostate-specific antigen, nerve growth factor γ, and epidermal growth factor-binding protein) and the activators for the kringle family proteins (including the human tissue plasminogen activator and human hepatocyte growth factor activator). Importantly, all of the residues known to be crucial for substrate binding, specificity, and catalysis by the serine proteases are conserved in the predicted NES1 protein, suggesting that it may be a protease. An antipeptide antibody directed against a unique region of the NES1 protein (amino acids 120-137) detected a specific 30-kilodalton polypeptide almost exclusively in the supernatant of the mRNA-positive MECs, suggesting that NESt is a secreted protein. The 1.4-kb NES1 mRNA was expressed in several organs (thymus, prostate, testis, ovary, small intestine, colon, heart, lung, and pancreas) with highest levels in the ovary; a 1.1-kb transcript was found in the pancreas. Although expression of the NES1 mRNA was observed in all normal and immortalized nontumorigenic MECs, the majority of human breast cancer cell lines showed a drastic reduction or a complete lack of its expression. The structural similarity of NES1 to polypeptides known to regulate growth factor activity and a negative correlation of NES1 expression with breast oncogenesis suggest a direct or indirect role for this novel protease-like gene product in the suppression of tumorigenesis.

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