Retroviral insertional mutagenesis identifies oncogene cooperation

Abstract

Retroviral insertional mutagenesis has been applied to identify oncogenes that are important for both human and rodent carcinogenesis. The method reveals not only primary oncogenes but also cooperative genes that might be affected as second or third hits in multistep carcinogenesis. The use of genetically engineered mice such as NUP98-HOXA9 transgenic mice enabled efficient identification of cooperative genes, which provides important information for the molecular pathway in carcinogenesis/leukemogenesis. With use of the retrovirus mediated gene transfer system, retroviral insertional mutagenesis will provide invaluable information to understand genetic interaction in complex mechanisms of carcinogenesis.

Figure 1

Mechanism of oncogene activation and tumor suppressor inactivation by retroviral insertional mutagenesis.

Figure 2

Molecular pathway of NUP98‐HOXA9 leukemogenesis. (A) NUP98‐HOXA9 promotes proliferation and self renewal of bone marrow cells, while Meis1 blocks differentiation of myeloid precursors (right panel). Analysis of individual tumors of NUP98‐HOXA9 tg/BXH2 leukemia divided CIS into two categories: overlapping and non‐overlapping genes with Meis1. The former gene such as Dnalc4 functions as an additional step for leukemogenesis like an antiapoptotic gene. On the other hand, the latter gene may replace Meis1 function. Cooperative activation between Hoxa7/a9 and Meis1 has also been observed in the wild type BXH2 leukemia (left panel). However, it is difficult to analyze molecular pathway due to less frequent Hox activation and interference of unrelated integration sites. (B) Pathway prediction by insertional mutagenesis. The upper panel indicates class of genes having equivalent function identified as integration sites. The bottom panel shows isolated genes belong to one particular molecular pathway.

Figure 3

Retroviral insertional mutagenesis using replication deficient retrovirus. (A) Co‐expression of the primary oncogene inserted into the retroviral vector and a cooperative in the vicinity of integration. (B) and (C) Peripheral blood smear of leukemic mice after bone marrow transfer. Transduction with the NUP98‐HOXA9 retrovirus induces chronic myeloproliferation (B) and eventually AML showing blastic cells (C). Giemsa. Original magnification, × 100. (D) Southern blot analysis of retrovirally induced NUP98‐HOXA9 leukemia. DNA samples were digested with BglII, gel electrophoresed and transferred onto nylon filter. The filter was probed with a DNA fragment of GFP that is inserted in the construct. Each band indicates clonal integration of the retroviral construct. (E) The NUP98‐HOXA9 retroviral integration at the Meis1 locus. The virus was inserted 6 kb upstream of the putative transcriptional start site of Meis1 with reverse transcriptional orientation.