Genomic mapping in outbred mice reveals overlap in genetic susceptibility for HZE ion- and γ-ray-induced tumors.

Cancer risk from galactic cosmic radiation exposure is considered a potential "showstopper" for a manned mission to Mars. Calculating the actual risks confronted by spaceflight crews is complicated by our limited understanding of the carcinogenic effects of high-charge, high-energy (HZE) ions, a radiation type for which no human exposure data exist. Using a mouse model of genetic diversity, we find that the histotype spectrum of HZE ion-induced tumors is similar to the spectra of spontaneous and γ-ray-induced tumors and that the genomic loci controlling susceptibilities overlap between groups for some tumor types. Where it occurs, this overlap indicates shared tumorigenesis mechanisms regardless of the type of radiation exposure and supports the use of human epidemiological data from γ-ray exposures to predict cancer risks from galactic cosmic rays.

Transformation of mortal human fibroblasts and activation of a growth inhibitory pathway by the bovine papillomavirus E5 oncoprotein.

The 44-amino acid bovine papillomavirus E5 protein induces tumorigenic transformation of immortal rodent fibroblasts by binding to and activating the platelet-derived growth factor beta receptor (PDGFbetaR). Here E5 was expressed in mortal human diploid fibroblasts (HDFs), which lack the accumulated genetic changes that are present in immortal rodent cells. E5 induced focus formation and morphological transformation of HDFs without inducing anchorage independence or immortalization. Similar effects were observed with the v-sis and neu* oncogenes. E5-PDGFbetaR complexes were observed in the E5-expressing HDFs, as was constitutive PDGFbetaR activation, which was required for the transforming activity of E5. The E5 HDFs attained a higher saturation density than the control cells, expressing increased levels of hyperphosphorylated retinoblastoma protein at subconfluent densities. However, when these cells reached confluence, growth inhibition accompanied by dramatic down-regulation of the PDGFbetaR, and retinoblastoma protein was induced apparently by a factor secreted into the medium. This may represent a novel negative feedback mechanism controlling PDGFbetaR-induced proliferation and thereby protecting against complete transformation.

Telomerase activation in human fibroblasts during escape from crisis.

The immortalization of human diploid fibroblasts requires the circumvention of both the senescence (M1) and crisis (M2) mechanisms of growth control. Cells expressing the SV40 T antigen virtually always bypass senescence, but only rarely escape crisis. The low frequency of this latter event indicates that cellular mutations are necessary to escape crisis. Thirteen subpopulations of T antigen-expressing human fibroblasts were cultured into crisis. Colonies that appeared to resume growth were assayed for telomerase activity, telomere maintenance, and the immortal phenotype. Our results show that 33 of 35 colonies were telomerase negative and were not immortal. Two colonies were telomerase positive when assayed in the first approximately 15 population doublings after crisis. The first was strongly positive, maintained telomeres at a stable short length, and was later determined to be immortal. The second initially had a weak telomerase signal, grew extremely slowly, and when examined had greatly elongated telomeres consistent with the ALT (alternative lengthening of telomeres) mechanism of telomere maintenance. These cells eventually grew faster and were later determined to be immortal. Additionally, two subpopulations had initially weak and later strong telomerase activity and the cells never entered a defined crisis period. We observed a perfect correlation between telomere maintenance and escape from crisis, supporting the hypothesis that the lack of stable telomeres causes crisis and that the ability to maintain telomeres abrogates crisis.

Identification of SV40 T-antigen mutants that alter T-antigen-induced chromosome damage in human fibroblasts.

The SV40 T antigen causes numerical (aneuploidy) and structural (aberrations) chromosome damage when expressed in human diploid fibroblasts. This chromosome damage precedes the acquisition of neoplastic traits such as anchorage independence, colony formation in reduced serum growth factors, immortalization, or tumorigenicity. Therefore, chromosome damage may be important in acquiring these traits because it could provide a mutational mechanism. To determine how the T antigen causes chromosome damage, point mutations were constructed that altered previously defined biochemical functions of the T protein. Mutant T antigen constructs were introduced into human diploid fibroblasts and selected by using G418. Clones of G418r cells that expressed mutant T antigens were expanded and scored for chromosome damage. Most of these mutant T antigens caused [corrected] levels of chromosome damage similar to those caused by [corrected] the wild-type T antigen. However, some T-antigen mutants induced fewer chromosome changes. A subset of these clones that induced less chromosome damage than wild-type T were examined further. Mutant T-antigen protein levels from this subset were quantified with flow cytometry and compared with wild-type protein expression levels. Mutations of T antigen shown previously to form less stable complexes with p53 caused less chromosome damage. A mutation in the zinc finger domain of T antigen also caused less chromosome damage. Interestingly, a mutant that caused loss of the ATPase activity of T antigen caused an increase in endoreduplicated cells. Also, a correlation was noted between cells expressing very low levels of T antigen (below detection limits when using flow cytometry) and an undamaged karyotype. This correlation indicates that there is a threshold level of T-antigen expression that induces chromosome damage and that expression levels on a per-cell basis rather than on a population basis should be considered in subsequent studies.

Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells.

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid fibroblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34(cdc2). These changes in mitotic control were evident within 5-10 population doublings after retroviral infection, indicating a direct effect of T expression. Cells acutely infected with the T-expressing retrovirus suffered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These findings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.

Telomerase activation during the linear evolution of human fibroblasts to tumorigenicity in nude mice.

The ribonucleoprotein enzyme telomerase is active in most immortal cell lines, most tumors and all tumor-derived cell lines. The enzyme is important because it prevents continual shortening of telomeres and therefore plays a significant role in chromosome maintenance. In man, telomerase is not active in most somal cells with finite lifespans. Using the SV40 T antigen we immortalized and transformed to fully tumorigenic a human fibroblast cell strain. We wished to determine when telomerase was activated during this progression to tumorigenicity. Using the PCR-based TRAP assay we found that eight of eight immortal cell lines that were either not tumorigenic or rarely formed tumors were telomerase positive at the time of inoculation. Additionally, 10 of 11 newly immortal cell lines contained telomerase activity within the first 25-33 population doublings after crisis. None of the precrisis cells from which these immortal cells were derived were positive for telomerase activity. Thus we found that telomerase activation is not the final in vivo step in the transformation of these cells and the window of activation is usually near the escape from crisis or M2. These results strengthen the hypothesis that telomerase activation may allow the rare cell to escape from crisis in those immortal cell populations dependent on telomerase for telomere maintenance.

Iterative chromosome mutation and selection as a mechanism of complete transformation of human diploid fibroblasts by SV40 T antigen.

The acquisition of an extended lifespan and of neoplastic properties, including anchorage independence, ability to grow in low serum-containing media, morphological transformation, immortalization and tumorigenicity in nude mice were studied in 31 human fibroblast lineages transfected with plasmids containing the SV40 early genes. Plasmids were used that contained sequences for large T alone, or large T plus small t or large T plus small t plus the SV40 origin. Cells expressing large T antigen gradually acquired the ability to form colonies in low serum or to form anchorage-independent colonies. Large T antigen was sufficient to cause complete transformation to tumorigenicity if multi-step lineage evolution was obtained by prolonged serial passage and if in vivo progression was assisted by means of a gelatin sponge implantation technique. Cells derived from progressive tumors initiated in sponges showed enhanced tumorigenicity as measured by ability to obtain tumors without using sponges and with reduced latent period, higher incidence and with fewer cells inoculated. Multiple lineages of human fibroblasts have been converted to tumorigenicity without additional treatments such as transfection with activated oncogenes or exposure to carcinogens. These data, taken in conjunction with earlier studies showing that T antigen causes chromosome mutation preceding and accompanying the accumulation of the neoplastic phenotype, suggests that the T protein drives the transformation process by acting as a mutagen and cells with growth advantages were selected for in vitro and in vivo. With the possible exception of morphological transformation, the presence or absence of genes for small t and the SV40 origin were not critical for the process.

SV40 T antigen induced chromosomal changes reflect a process that is both clastogenic and aneuploidogenic and is ongoing throughout neoplastic progression of human fibroblasts.

In human fibroblasts, the expression of SV40 large T antigen is known to cause a variety of chromosomal aberrations and especially dicentric chromosomes. In some cases, the later aberrations have been reported to be reversible telomeric associations. We report here aberration and chromosome number studies of twenty-nine T antigen positive lineages, studied from their initiation by transfection of T antigen sequences into human diploid fibroblasts, until crisis or immortalization occurred or, in some cases until the lines became tumorigenic in nude mice. The data show that T antigen consistently produced chromosomal instability of both number and structure by an active process that began before transformation indicators were positive and continued throughout neoplastic progression. The most frequently observed aberrations were dicentric chromosomes, which were shown to be true dicentrics by examination by in situ hybridization with telomeric sequences. These data are consistent with the hypothesis that T antigen causes human fibroblasts to become neoplastically transformed by successive rounds of chromosomal mutation and lineage evolution.

Frequent deletions in nine newly immortal human cell lines.

Nine newly immortal lines of human fibroblasts transfected with SV40 T antigen were examined for recurrent chromosome losses. In order of decreasing frequency, all nine lines had three or more of the following minimal deletions specifically associated with the immortalization event: del(6)(q21), del(3)(p24), del(1)(p34), del(4)(p25), del(5)(p14), del(11)(p11), del(11)(q14), del(12)(p12), and del(14)(p?). Many other chromosome changes were not clearly associated with immortalization, but were acquired during other stages of this multistep model of neoplastic transformation. We propose that these chromosome loci associated with immortalization are candidates for the location of genes involved in cellular senescence.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family Falconidae.

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

Spontaneous in vitro neoplastic evolution: selection of specific karyotypes in Chinese hamster cells.

Recurrent cytogenetic changes occurred reproducibly in vitro during the spontaneous neoplastic evolution of cultured Chinese hamster cells. In particular, excess 3q material appeared shortly after immortalization in numerous independent trials. By contrast, when clones were isolated at the earliest possible time after immortalization, a wide spectrum of types of cytogenetic evolution followed, which also resulted in transformed and tumorigenic cells. Clones with stable distinct colonial morphologies were used to demonstrate growth rate interactions when subpopulations compete. We conclude that specific recurring karyotypes are associated with specific stem lines with transient growth advantage during the early stages of in vitro carcinogenesis. Stem lines with other karyotypic change or no detectable karyotypic change are almost equally capable of undergoing the entire spontaneous neoplastic process in vitro.

Karyotype instability of Chinese hamster cells during in vivo tumor progression.

The extent of karyotype instability in spontaneously transformed Chinese hamster cells was determined after tumor formation by cytogenetic analysis of karyotype heterogeneity. The degree of karyotype heterogeneity among tumors formed in nude mice correlated with tumor latent period. The karyotypes of tumors formed after a short latent period by cells of high tumorigenic potential were similar to each other and to the injected cells. The karyotypes of tumors from cells of low tumorigenic potential and long latents periods were diverse, however. No chromosome aberration was common to every tumor. These results suggest that preneoplastic cells whose phenotypes are not directly capable of tumor formation can progress in vivo and that karyotype instability plays an important role in providing cell variants for tumor progression.

Spontaneous immortalization rate of cultured Chinese hamster cells.

Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation. The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity. Immortality was not related to rare, precommitted cells from the animals. The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells. On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal. In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization. This analysis yielded a rate of 1.9 X 10(6) per cell per generation.