Friederike C. von Lintig

Ras activation in human breast cancer

Genetic ras mutations are infrequent in breast cancer but Ras may be pathologically activated in breast cancer by overexpression of growth factor receptors which signal through Ras. Using a highly sensitive, coupled enzymatic assay, we measured Ras activation in 20 breast cancers, two fibroadenomas, and seven normal breast samples. Ras was highly activated compared to benign tissue in 11 of the 20 cancer; 7 of these 11 cancers expressed both the epidermal growth factor (EGF) and ErbB-2/neu/HER-2 receptors with the remaining four cancers with high Ras activation expressing one of these two receptors. In the other nine cancers, Ras activation was similar to that observed in benign breast tissue with none of these cancers expressing the EGF receptor while one expressed the ErbB-2 receptor. None of the cancers tested had an activating K-ras mutation nor did any of the cancers express a truncated EGF receptor or the c-FMS receptor. The activity of mitogen-activated protein (MAP) kinase was high in the cancers, and reflected the degree of Ras activation. In cultured mammary tumor cell lines, we showed that Ras activation was ligand dependent in cells overexpressing the ErbB-2 receptor. Thus, Ras was abnormally activated in breast cancers overexpressing the EGF and/or ErbB-2 receptors indicating there are sufficient ligands in vivo to activate these receptors, and this work provides a basis for new target-based treatments of this disease.

Rheb is in a high activation state and inhibits B-Raf kinase in mammalian cells

Rheb (Ras homolog enriched in brain) is a member of the Ras family of proteins, and is in the immediate Ras/Rap/Ral subfamily. We found in three different mammalian cell lines that Rheb was highly activated, to levels much higher than for Ras or Rap 1, and that Rhebs activation state was unaffected by changes in growth conditions. Rhebs high activation was not secondary to unique glycine to arginine, or glycine to serine substitutions at positions 14 and 15, corresponding to Ras residues 12 and 13, since Rheb R14G and R14G, S15G mutants had similarly high activation levels as wild type Rheb. These data are consistent with earlier work which showed that purified Rheb has similar GTPase activity as Ras, and suggest a relative intracellular deficiency of Rheb GTPase activating proteins (GAPs) compared to Rheb activators. Further evidence for relatively low intracellular GAP activity was that increased Rheb expression led to a marked increase in Rheb activation. Rheb, like Ras and Rap1, bound B-Raf kinase, but in contrast to Ras and Rap 1, Rheb inhibited B-Raf kinase activity and prevented B-Raf-dependent activation of the transcription factor Elk-1. Thus, Rheb appears to be a unique member of the Ras/Rap/Ral subfamily, and in mammalian systems may serve to regulate B-Raf kinase activity.

Cell Type-specific Regulation of B-Raf Kinase by cAMP and 14-3-3 Proteins

Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.

Activation of the Rap1 Guanine Nucleotide Exchange Gene,CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia

Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factorCalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 13278–13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show thatCalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification ofCalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia.

Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice

We have previously documented that transgenic mice expressing SV40 Tag regulated by the rat prostatic steroid-binding protein C3(1) 5′-flanking region display multistage mammary tumorigenesis. To delineate genetic changes associated with mammary tumor progression, comparative genomic hybridization (CGH) was performed. CGH revealed a consistent gain of the telomeric region of chromosome 6. This region contains the Ki-ras proto-oncogene. Analyses of genomic DNA by Southern blot demonstrated up to 40-fold amplification of the Ki-ras gene. Ki-ras amplification was detected in 12, 46 and 68% of tumors from 4, 5 and 6 month old mice, respectively, whereas no amplifications were found in any preneoplastic mammary tissues. Tumors bearing Ki-ras gene amplification exhibited high levels of Ki-ras RNA and protein. The over-expressed Ki-Ras protein in these tumors appeared functionally active as indicated by the elevated MAP kinase activity. These data demonstrate that while Ki-ras amplification might not be an early event, there is a strong association between Ki-ras amplification and over-expression and mammary tumor progression in this model. This study also shows that CGH is a powerful and useful technique for identifying chromosomal copy number changes during tumor progression, and that this model may provide a predictable in vivo system for studying gene amplification.

Quantitative determination of Rap 1 activation in cyclic nucleotide-treated HL-60 leukemic cells: lack of Rap 1 activation in variant cells

We have previously isolated variant HL-60 cells that are resistant to cGMP-induced differentiation and showed that they are deficient in proteolytic cleavage and/or carboxyl methylation of Rap 1A (J. Biol. Chem. 269, 32155–32161, 1994 and Oncogene 17, 2211–2233, 1998). We have now developed an enzyme-based method for assessing Rap 1 activation which is quantitative and provides a measurement of the per cent of Rap molecules in the active GTP-bound state. Using this method, we show that cAMP and cGMP analogs activate Rap 1 in parental HL-60 cells but not in the variant cells and that H-89, a cAMP-dependent protein kinase inhibitor, has no effect on cAMP-induced Rap 1 activation in parental cells. Thus, cAMP activation of Rap 1 in HL-60 cells is likely through a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) and since cAMP does not activate Rap 1 in the variant cells, the data suggest that full post-translational processing of Rap 1 is necessary for cAMP-GEF activation of Rap 1. Activation of Rap 1 by cGMP analogs has not been previously found and suggests possible cross-talk between the NO/cGMP signal transduction pathway and Rap 1 signaling.

Haploid loss of Ki-ras delays mammary tumor progression in C3 (1)/SV40 Tag transgenic mice.

We have previously demonstrated that amplification and overexpression of the Ki-ras gene is associated with mammary tumor progression in C3(1)/SV40Tag transgenic mice (Liu et al., 1998). To further evaluate the functional significance of the Ki-ras proto-oncogene in mammary cancer development, in vivo studies were conducted to examine the effect of Ki-ras gene dosage on tumor progression. The lack of one normal Ki-ras allele C3(1)/SV40Tag transgenic mice resulted in significantly delayed mammary intraepithelial neoplasia (MIN) formation as well as in a decreased number of mammary gland carcinomas. However, despite the retardation of tumor development by reduced Ki-ras gene dosage, overall survival was only modestly affected. This appears to be due to several factors including significant mammary tumor growth associated with Ki-ras gene amplification and over-expression that occurs during the advanced stage of oncogenesis in mice carrying either one or two normal Ki-ras alleles. The retardation of tumor progression due to the haploid loss of Ki-ras did not appear to be related to accelerated apoptosis, or a reduced rate of cell proliferation at the tumor stages examined. These data strongly suggest that the gene dosage of Ki-ras affects tumor promotion at an early stage of mammary tumor progression in this SV40 Tag-induced model of mammary oncogenesis.

1,25-Dihydroxyvitamin D3 but not TPA activates PLD in Caco-2 cells via pp60c-src and RhoA

In the accompanying paper [Khare et al., Am. J. Physiol. 276 ( Gastrointest. Liver Physiol. 39): G993-G1004, 1999], activation of protein kinase C-α (PKC-α) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60c- src have been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)2D3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)2D3, but not TPA, activated pp60c- src and treatment with PP1, a specific inhibitor of the pp60c- src family, blocked the ability of 1,25(OH)2D3to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60c- src and PKC-α by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3or inhibit the stimulation of PKC-α by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3and TPA each activate PLD in Caco-2 cells in part via PKC-α, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60c- src and RhoA in the activation of PLD by 1,25(OH)2D3, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)2D3and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.