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

Abstract

We have previously documented that transgenic mice expressing SV40 Tag regulated by the rat prostatic steroid-binding protein C3(1) 5'-¯anking 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 ampli®cation of the Kiras gene. Ki-ras ampli®cation was detected in 12, 46 and 68% of tumors from 4, 5 and 6 month old mice, respectively, whereas no ampli®cations were found in any preneoplastic mammary tissues. Tumors bearing Ki-ras gene ampli®cation 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 ampli®cation might not be an early event, there is a strong association between Ki-ras ampli®cation 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 ampli®ca-tion.

The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma

“…Comparative genomic hybridization has revealed that mammary tumor progression in the C3(1)/Tag transgenic mice is associated with a gain of genomic material from the distal region of mouse chromosome 6 (Liu et al, 1998). We have demonstrated by Southern blot analyses that the ki-ras gene, which is contained within this region of chromosome 6, is ampli®ed up to 30-fold.…”

“…We have demonstrated by Southern blot analyses that the ki-ras gene, which is contained within this region of chromosome 6, is ampli®ed up to 30-fold. Furthermore, the amplification of ki-ras correlates with increased expression of ki-ras RNA and protein and is associated with increased MAP kinase activity (Liu et al, 1998). In order to determine whether the ampli®cation and overexpression of ki-ras is functionally important for tumor progression in this model, mice carrying the C3(1)/Tag transgene and lacking one allele of ki-ras were generated and studied.…”

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

“…To measure Rapbound GTP, we used the RBD peptide to precipitate Rap´GTP from cell extracts and we measured GTP eluted from Rap using an enzymebased method (Scheele et al, 1995;Pilz et al, 1997;Guha et al, 1996Guha et al, , 1997Prigent et al, 1996;Liu et al, 1998;Egawa et al, 1999;Sharma et al, 1998). To determine the sum of Rap-bound GTP and GDP, we converted Rap´GDP to Rap´GTP by incubating half of the cell extract with 10 mM GTP and then measured Rap´GTP as described above.…”

“…In experiments where BHK cells were transfected with GST-tagged Rap 1A, the sum of GTP plus GDP bound to Rap 1A was measured by converting GDP to GTP using the enzyme pyruvate kinase (Sharma et al, 1998;Liu et al, 1998;Egawa et al, 1999) and total GTP was then measured.…”

Centrosome abnormalities, recurring deletions of chromosome 4, and genomic amplification of HER2/neu define mouse mammary gland adenocarcinomas induced by mutant HER2/neu

“…For instance, the forced expression of c-myc, HER2/neu, and cyclinD1, respectively, under the control of the mammary gland speci®c MMTV-promoter results in mammary gland adenocarcinomas (Muller et al, 1988;Stewart et al, 1984;Wang et al, 1994). Accordingly, the conditional, tissue speci®c depletion of BRCA1 function results in tumor formation (Xu et al, 1999a,b) and the impairment of p53 and RB1 function via expression of SV40 large T antigen under the control of the C3 promoter induces K-ras ampli®cation and tumorigenesis (Liu et al, 1998). As in human cancers, it is likely that tumorigenesis in the mouse is promoted, or at least accompanied by, the acquisition of non-random chromosomal aneuploidies.…”