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Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.

Overexpression of c-raf-1 and the myc family of protooncogenes is primarily associated with small cell carcinoma, which accounts for approximately 25% of human lung cancer. To determine the functional significance of the c-raf-1 and/or c-myc gene expression in lung carcinogenesis and to delineate the relationship between protooncogene expression and tumor phenotype, we introduced both protooncogenes, alone or in combination, into human bronchial epithelial cells. Two retroviral recombinants, pZip-raf and pZip-myc, containing the complete coding sequences of the human c-raf-1 and murine c-myc genes, respectively, were constructed and transfected into simian virus 40 large tumor antigen-immortalized bronchial epithelial cells (BEAS-2B); this was followed by selection for G418 resistance. BEAS-2B cells expressing both the transfected c-raf-1 and c-myc sequences formed large cell carcinomas in athymic nude mice with a latency of 4-21 weeks, whereas either pZip-raf- or pZip-myc-transfected cells were nontumorigenic after 12 months. Cell lines established from tumors (designated RMT) revealed the presence of the cotransfected c-raf-1 and c-myc sequences and expressed morphological, chromosomal, and isoenzyme markers, which identified BEAS-2B cells as the progenitor line of the tumors. A significant increase in the mRNA levels of neuron-specific enolase was detected in BEAS-2B cells containing both the c-raf-1 and c-myc genes and derived tumor cell lines. The data demonstrate that the concomitant expression of the c-raf and c-myc protooncogenes causes neoplastic transformation of human bronchial epithelial cells resulting in large cell carcinomas with certain neuroendocrine markers. The presented model system should be useful in studies of molecular events involved in multistage lung carcinogenesis.