Studies in cell culture have suggested that the level of RAS expression can influence the transformation of cells and the signaling pathways stimulated by mutant RAS expression. However, the levels of RAS expression in vivo appear to be subject to feedback regulation, limiting the total amount of RAS protein that can be expressed. We utilized a bitransgenic mouse lung tumor model that expressed the human Ki-ras(G12C) allele in a tetracycline-inducible, lung-specific manner. Treatment for 12 months with 500 mu g/ml of doxycycline (DOX) allowed for maximal expression of the human Ki-ras(G12c) allele in the lung, and resulted in the development of focal hyperplasia and adenomas. We determined if different levels of mutant RAS expression Would influence the phenotype of the lung lesions. Treatment with 25, 100 and 500 mu g/ml of DOX resulted in close-dependent increases in transgene expression and tumor multiplicity. Microscopic analysis of the lungs of mice treated with the 25 mu g/ml dose of DOX revealed infrequent foci of hyperplasia, whereas mice treated with the 100 and 500 mu g/ml doses exhibited numerous hyperplastic foci and also adenomas. Immunohistochemical and RNA analysis of the downstream effector pathways demonstrated that different levels of mutant RAS transgene expression resulted in differences in the expression and/or phosphorylation of specific signaling molecules. Our results suggest that the molecular alterations driving tumorigenesis may differ at different levels of mutant Ki-ras(G12C) expression, and this should be taken into consideration when inducible transgene systems are utilized to promote tumorigenesis in mouse models. (c) 2008 Elsevier Inc. All rights reserved.

Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites.

The histologic diagnosis of tumors based upon tissue architectural changes, cytologic examination of cells, and immunohistochemical reactions is the mainstay of any surgical pathology laboratory. However, a wide variety of cytogenetic and molecular assays not only provides extremely usefill information regarding patient diagnosis but also carries valuable prognostic and therapeutic inlplications. Cytogenetic data are particularly applicable to the diagnosis of a myriad of hematologic malignancies and solid tumors. Here, we describe a variety of classic and molecular cytogenetic techniques that are used routinely in the diagnosis of these tutnors, with a focus on classical chromosomal analysis and fluorescence in situ hybridization. In addition, spectral karyotyping, comparative genomic hybridization, and array technology are discussed. (The J Histotechnol 28:209, 2005)Submitted November 9, 2005; accepted with revisions Decelnber 1, 2005

Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CT alpha prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CT alpha as a negative regulator of polyploid formation.