Suppression of tumorigenicity of rat liver epithelial tumor cell lines by a putative human 11p11.2-p12 liver tumor suppressor locus.

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of a rat liver tumor cell line (termed GN6TF) which contains well defined chromosomal aberrations involving rat chromosomes 1, 4, 7, and 10. In the present study, we investigated the potential of this human 11p11.2-p12 liver tumor suppressor locus to suppress the tumorigenic potential of two other rat liver tumor cell lines (GN3TG and GP10TA) following microcell-mediated introduction of human chromosome 11. These tumor cell lines are aneuploid and contain chromosomal abnormalities that are similar to the GN6TF tumor line. The tumorigenic potential and other phenotypic characteristics of GN3TG-11neo and GP10TA-11neo microcell hybrid (MCH) cell lines were variable, and dependent upon the status of the introduced human chromosome 11. MCH cell lines that retained the region of 11p11. 2-p12 delineated by microsatellite markers D11S1385 and D11S903 exhibited suppression of tumorigenicity in vivo (decrease in tumorigenicity and/or elongation of latency), whereas, the tumorigenic potential of one MCH line that lacked markers in this region of human 11p11.2-p12, but retained flanking markers, was not changed from that of the parental tumor cell line. The chromosomal interval between microsatellite markers D11S1385 and D11S903 encompasses the previously localized minimal liver tumor suppressor region, suggesting that a common locus is responsible for tumor suppression among the rat liver tumor cell lines examined. The results of the present study have verified the presence of a liver tumor suppressor locus within human 11p11.2-p12, and have identified a substantial number of microsatellite markers that are closely linked to this tumor suppressor region. These chromosomal markers will facilitate positional cloning of candidate genes from this region, and may prove useful for determining the involvement of this locus in the pathogenesis of human liver cancer.

Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells.

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.

Long-term episomal gene delivery in human lymphoid cells using human and avian adenoviral-assisted transfection.

Human B-lymphoblastoid cells transformed by Epstein-Barr virus (EBV), a common source of established human cell lines, are generally refractory to standard DNA transfection methods. We report here the use of human and chicken adenoviruses for the stable delivery of plasmid DNA carrying the latent origin of replication, oriP, from EBV into human B-lymphoblastoid cells. Long-term hygromycin-resistant transformants of human lymphoblastoid cells belonging to the genetically unstable inherited disease Fanconi's anemia were obtained by adenoviral-assisted transfection of plasmid DNA complexed to either human adenovirus type 5 (Ad5) or avian adenovirus type 1 (CELO). Successful long-term transformation of human lymphoblastoid cells was unrelated to the efficiency of short-term transfection as measured by the transient expression level of a reporter lacZ gene. The human or avian adenoviral binary conjugates, with no other cell ligand, were sufficient for stable cell transformation. The amount of DNA established in the human B-cell transformants was approximately 100-fold higher with the CELO virus. We conclude that Ad5-assisted transfection is more suitable for transient gene expression studies in human lymphoblastoid cells, while CELO-assisted transfection appears the method of choice for stable genetic transformation of such cells. Such differential short- and long-term gene delivery strategy with Ad5 and CELO may be useful in gene therapy of human B cells involving cancers and inherited diseases, respectively.