The mouse mammary tumor virus (MMTV) is a potent somatic mutagen, integrating within the host genome and disrupting normal gene expression. One of these genes encodes the p48 component of eucaryotic intiation factor 3 (EIF3P48/INT6). The role of EIF3P48/INT6 in human carcinogenesis is unclear. Recently, Nupponen et al. (42) have shown that the gene encoding another EIF3 subunit, p40; is amplified and overexpressed in breast and prostate carcinomas. This observation coupled with the rearrangement of eIF3p48/Int6 by MMTV in mouse mammary tumors raised the possibility that the human EIF3P48/INT6 gene may also be a target for mutation in certain human malignancies. To investigate this possibility EIF3P48/INT6 was examined for genetic abnormalities in two of the most common forms of cancer in humans, breast (n=62) and non-small cell lung (n=78) (NSCLC) carcinomas. Loss of heterozygosity (LOH) at EIF3P48/INT6 was detected at 5 (24%) of 24 informative breast tumors and 10 (29%) of 34 informative lung tumors. In those tumors having LOH no point mutations were found by SSCP analysis in the remaining allele. Loss of EIF3P48/INT6 expression was observed in 23 (37%) of breast tumors and 24 (31%) of NSCLC. This was not related to commonly used pathological parameters in breast cancer patients, while in NSCLC patients loss of EIF3P48/INT6 expression was mainly seen in adenocarcinomas (p<0.0001). Additional studies on larger series of tumor specimens with long term follow-up are needed to determine whether EIF3P48/INT6 expression may represent a new prognostic or predictive marker. To begin to understand how expression of truncated eIF3p48/Int6 contributes to mammary gland tumorigenesis, I have used the yeast 2-hybrid system to screen a mouse cDNA library for genes that encode proteins that interact with EIF3P48/INT6. One such gene (DOC1R) is related to the human Deleted in Oral Cancer-1 (DOC1) gene that is involved in TNF-a mediated apoptosis, and cell cycle control through specific interactions with CDK2 and DNA polymerase a/primase.The DOC1 and DOC1R proteins overall are 57% identical whereas the C-terminal 52 residues are 86% identical. The specificity of the interaction of murine Doc1R and EIF3P48/INT6 was confirmed by an in vitro """"""""pull down"""""""" experiment, in which tagged DOC1R protein was reacted with a 35S Met labeled EIF3P48/INT6 in vitro translation product. DOC1R is ubiquitously expressed as 1.2 kb RNA species in mouse tissues. The predicted protein is 13 KDa and is localized in the cytoplasm and nucleus. Using Drosophila EIF3P48/INT6, we performed a yeast 2-hybrid screen of an adult Drosophila cDNA library to isolate proteins that interact with full-length Drosophila EIF3P48/INT6. Out of 7 x 107 clones tested, 28 cDNA clones were isolated that encode a novel protein designated D6BP (Drosophila Int6 binding protein). The corresponding prey protein encoded by the D6BP gene was shown to bind strongly and specifically to full length, but not truncated, Drosophila eIF3p48/Int6 by growth on selective media and ?-galactosidase expression of yeast transformants. It is conserved in Drosophila, human, rabbit, dog, chicken, and mouse. D6BP is expressed as a 4.0 Kb RNA transcript throughout all stages of development from the 0-4 hour embryo through the adult. The C-terminal 60 amino acids of D6BP are sufficient for binding to EIF3P48/INT6. Although D6BP does not correspond to any known gene, amino acid sequence analysis suggests that D6BP may be a transcription factor. It contains nuclear localization signals, a PHD finger domain, and shares 42% amino acid similarity over 130 amino acids with the Drosophila ASH-1 protein, a member of the trithorax family. Additionally, the predicted D6BP protein sequence shares 38% amino acid similarity over 905 amino acids with the human TZP (tudor domain, zinc finger, PHD finger) transcription factor.
