Constitutive modulation of Raf-1 protein kinase is associated with differential gene expression of several known and unknown genes.
BACKGROUND
Raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Consistent with this notion is the fact that constitutive modulation of expression and/or activity of Raf-1 protein kinase modifies cell growth, proliferation, and cell survival. Although these effects are controlled at least in part by transcriptional mechanisms, the role of Raf-1 in the regulation of specific gene expression is unclear.


METHODS
Differential display of mRNA was used to identify the genes differentially expressed in human head and neck squamous carcinoma cells (PCI-06A) transfected with either the antisense c-raf-1 cDNA (PCI-06A-Raf(AS)), or a portion of cDNA coding for the kinase domain of Raf-1 (PCI-06A-Raf(K)). The differentially expressed fragments were cloned and sequenced, and they were used as probes to compare the expression patterns in parent transfectants by Northern blot analysis. In addition, expression patterns of the novel genes were examined in normal tissues and cancer cell lines.


RESULTS
Six differentially expressed cDNA fragments were identified and sequenced. Northern blot analysis revealed that four of these fragments representing human alpha 1-antichymotrypsin (alpha 1-ACT), mitochondrial cytochrome c oxidase subunit II (COX-II), and two as-yet unidentified cDNAs (KAS-110 and KAS-111) were relatively overexpressed in PCI-06A-Raf(AS) transfectants compared with PCI-06A-Raf(K) transfectants. The other two cDNA fragments representing human elongation factor-1 alpha (HEF-1 alpha) and ornithine decarboxylase antizyme (OAz) were overexpressed in PCI-06A-Raf(K) transfectants compared with PCI-06A-Raf(AS) transfectants. The KAS-110 (114 bp) and KAS-111 (202 bp) cDNAs did not show significant matches with sequences in the GenEMBL, TIGR, and HGS DNA databases, and these may represent novel genes. The KAS-110 and KAS-111 transcripts, approximately 0.9 kb and approximately 0.5 kb, were observed in most normal tissues and several cancer cell types, indicating their housekeeping function.


CONCLUSIONS
This study reports novel components of the Raf-1 signaling pathway. alpha 1-ACT, HEF-1 alpha, COX-II, and OAz have been previously implicated in diverse cellular responses including transformation, energy metabolism, and cell survival. Our data suggest that expression of these genes may play a role in the Raf-1-mediated biological activity of PCI-06A cells. The KAS-110 and KAS-111 cDNAs represent unknown genes, and further investigations are necessary to determine their role in the cellular response. Identification of specific targets may provide useful markers for prognosis and therapy selection in squamous cell carcinoma.
Patel
S
S
Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007, USA.
Wang
F H
FH
Whiteside
T L
TL
Kasid
U
U
D001483:Base Sequence; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf