Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells.

Abstract

Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.Rapid advances in understanding the molecular changes in human oncogenesis have been provided by transfection of tumor DNAs into NIH 3T3 cells (1). Of all human tumor DNA tested, however, some 70% are ineffective in 3T3 transformation (2). In developing a DNA transfection system using normal human cells as recipients (3), we found that the cells could be transformed to anchorage-independent growth by MOLT-4 DNA, which transforms 3T3 cells inefficiently (4) or not at all (5). We have used a series of three multiple myeloma cell lines, GM 1312, GM 1500, and GM 2132, to examine transformation of human cells by tumor DNAs that do (GM 1500 and GM 2132) and do not (GM 1312) transform NIH 3T3 cells (5).We show that DNA of GM 1312 does transform human cells to anchorage-independent growth, yielding transfectants that grow in soft agar at much higher frequencies than do the parental fibroblasts and whose DNA can transform naive recipients to anchorage-independent growth. DNAs of GM 2132 and GM 1500 transform human cells at a higher frequency than does that from GM 1312. Since GM 1500 DNAs transform both human and 3T3 cells, we tested by restriction analysis whether the same gene was acting in the two systems: although restriction by five enzymes gave similar results in the two systems, Sac I inactivated the transforming activity of the DNA in 3T3 cells (5) but not in human cells, suggesting the possibility that different genes are recognized by the two cellular recipients.These results show that the human cell transfection system can detect transforming genes in DNAs ineffective in the 3T3 system and indicate the possibility of detecting additional transforming sequences in DNAs containing oncogenes that can transform 3T3 cells. MATERIALS AND METHODSCells. Neonatal human fibroblasts were obtained from primary cultures established in this laboratory with foreskins from Central Suffolk Hospital (Riverhead, NY) or Brookhaven Hospital (Patchogue, NY). Stock cultures were grown without antibiotics in a Dulbecco's modified Eagle's m...

Oncogenic Transformation by Cellular DNA Isolated from Human Cytomegalovirus-lnfected Cells

“…It is possible that £coRI inactivates a gene or genes (or enhancer-pro moter sequences) which is (are) responsible for the oncogenic transformation process [26,28]. These findings are consistent with previous re ports [33,40,41] which demonstrated that the transforming ability of DNA extracted from different human malignancies was selectively sensitive to restriction enzyme digestion. Al though it may only be coincidence, it is interes ting to note that restriction enzymes Xba\ or ///« d ill leave the transforming region of AD-169 intact, whereas digestion with EraRI inacti vates this function [3,19,20].…”

“…CMV infection has been shown to result in extensive changes at the DNA [23,24] and chromosomal levels [23,25,39] which con ceivably could be related to the increased trans forming activity of the cellular DNA. The cal culated efficiency obtained for the transfor ming activity of cellular DNA (foci/pg DNA) from CMV-infected cells is similar to the focus forming activity of HMW DNA isolated from 70 Boldogh/Huang/Baskar/Gergely/ Albrecht Transformation by CMV-Altercd Cellular DNA different human tumor biopsies [33,40,41], From day 12 after exposure, the focus-forming activity of cellular DNA decreased and reached an undetectable level by day 20. The loss of transforming activity of HMW DNA at this stage of infection may be related to the extensive changes occurring in the nucleus (e.g., condensation of nuclear content and for mation of nuclear inclusions) [29] and the ex tensive degradation of cellular DNA apparent during the late phase of CMV replication [Boldogh et al, unpubl.…”

Cell Cycle Regulator GeneCDC5L, a Potential Target for 6p12-p21 Amplicon in Osteosarcoma

“…These tumors were extremely slow growing. Such tumors arising from NIH3T3 cells injected to severe combined immunodeficient mice was previously been reported (29). Mice were sacrificed at 10 wk following injection and tumors were harvested.…”

Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

“…Several variations of the assay have been used in attempts to detect other oncogenes, including the use of other recipient cell lines (3,12,17,33), assays for tumor formation in nude mice rather than transformation in cell culture (39), and even attempts to activate DNA from normal cells by adding strong promoter elements before DNA transfer. We have reported that a hamster embryonic cell line, SHOK (Syrian hamster Osaka Kanazawa), can be used as a good alternative to NIH 3T3 cells (14).…”