Differential expression of elongation factor-2, alpha4 phosphoprotein and Cdc5-like protein in...

The differential display of mRNAs technique was used to identify genes which are differentially expressed in the prolactin (PRL)-dependent Nb2-11C and PRL-independent Nb2-Sp rat lymphoma cell lines. The technique was validated by the differential display of the proto-oncogene c-myc in PRL-treated, but not in untreated, Nb2-11C cells. Nine DNA bands, isolated from the 6% display gels and confirmed by reverse Northerns to be differentially expressed, were cloned and gave rise to ten unique clones. DNA sequencing showed that these clones had limited homologies to known genes or uncharacterized expressed sequence tags. Using one of these clones (6ac1.12) as a probe in Northern analysis, a transcript of approximately 4 kb was detected which was elevated in PRL-treated Nb2-11C cells and in mid-log phase growing Nb2-Sp cells. Similar to c-myc, expression of the 4 kb transcript increased in Nb2-11C cells given PRL for 3 h (+/- the phorbol ester TPA) but not in cells given TPA alone. The 4 kb transcript also increased with increasing Nb2-11C cell densities. By screening an Nb2-Sp cDNA library with 6ac1.12 as probe, three unique genes were isolated and identified as elongation factor 2 (EF-2), alpha4 phosphoprotein and a Cdc5-like protein. Each of the three genes were PRL responsive in Nb2-11C cells and expressed constitutively in Nb2-Sp cells. The expression of EF-2 or alpha4, but not the Cdc-like protein, was dependent on cell densities. EF-2 regulates protein synthesis while the alpha4 and Cdc5-like phosphoproteins have been implicated in IgG receptor-mediated and mitogen-activated signaling, respectively. The identification that these genes are PRL-responsive and/or differentially expressed in the Nb2-11C and Nb2-Sp cell lines may permit insights into the molecular changes that are involved in regulating the transition to growth factor independence in lymphoid tumors.