Sarah M. Planchon

Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation

Regulation of transcriptional responses in growth‐arrested human cells under conditions that promote potentially lethal damage repair after ionizing radiation (IR) is poorly understood. Sp1/retinoblastoma control protein (RCP) DNA binding increased within 30 min and peaked at 2–4 h after IR (450–600 cGy) in confluent radioresistant human malignant melanoma (U1‐Mel) cells. Increased phosphorylation of Sp1 directly corresponded to Sp1/RCP binding and immediate‐early gene induction, whereas pRb remained hypophos‐phorylated. Transfection of U1‐Mel cells with the human papillomavirus E7 gene abrogated Sp1/RCP induction and G0/G1 cell cycle checkpoint arrest responses, increased apoptosis and radiosensitivity, and augmented genetic instability (i.e., increased polyploidy cells) after IR. Increased NF‐κB DNA binding in U1‐Mel cells after IR treatment lasted much longer (i.e., >20 h). U1‐Mel cells overexpressing dominant‐negative IκBα S32/36A mutant protein were significantly more resistant to IR exposure and retained both G2/M and G0/G1 cell cycle checkpoint responses without significant genetic instability (i.e., polyploid cell populations were not observed). Nuclear p53 protein levels and DNA binding activity increased only after high doses of IR (>l200 cGy). Disruption of p53 responses in U1‐Mel cells by E6 transfection also abrogated G0/G1 cell cycle checkpoint arrest responses and increased polyploidy after IR, but did not alter radiosensitivity. These data suggest that abrogation of individual components of this coordinate IR‐activated transcription factor response may lead to divergent alterations in cell cycle checkpoints, genomic instability, apoptosis, and survival. Such coordinate transcription factor activation in human cancer cells is reminiscent of prokaryotic SOS responses, and further elucidation of these events should shed light on the initial molecular events in the chromosome instability phenotype.—Yang, C.‐R., Wilson‐Van Patten, C., Planchon, S. M., Wuerzberger‐Davis, S. M., Davis, T. W., Cuthill, C., Miyamoto, S., Boothman, D. A. Coordinate modulation of Sp1, NF‐kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. FASEB J. 14, 379–392 (2000)

Growth factor-dependent AKT activation and cell migration requires the function of c-K(B)-Ras versus other cellular ras isoforms.

K-Ras-negative fibroblasts are defective in their steady-state expression of MMP-2. This occurs through c-K(B)-Ras dependent regulation of basal levels of AKT activity. In this report, we have extended those studies to demonstrate that in the absence of K-Ras expression, PDGF-BB fails to induce significant AKT activation, although this was not the case in N-Ras-negative cells. This phenotype was directly linked to PDGF-dependent cell migration. All of the independently immortalized K-Ras-negative cells failed to migrate upon the addition of PDGF. Only ectopic expression of c-K(B)-Ras, not c-K(A)-Ras nor oncogenic N-Ras, could restore both PDGF-dependent AKT activation and cell migration. Since most Ras binding partners can interact with all Ras isoforms, the specificity of PDGF-dependent activation of AKT and enhanced cell migration suggests that these outcomes are likely to be regulated through a c-K(B)-Ras-specific binding partner. Others have published that of the four Ras isoforms, only K(B)-Ras can form a stable complex with calmodulin (CaM). Along those lines, we provide evidence that 1) PDGF addition results in increased levels of a complex between c-K(B)-Ras and CaM and 2) the biological outcomes that are strictly dependent on c-K(B)-Ras (AKT activation and cell migration) are blocked by CaM antagonists. The PDGF-dependent activation of ERK is unaffected by the absence of K(B)-Ras and presence of CaM antagonists. This is the first example of a linkage between a specific biological outcome, cell migration, and the activity of a single Ras isoform, c-K(B)-Ras.