Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.

Selective killing of preneoplastic and neoplastic cells by methotrexate with leucovorin

Three sublines of NIH 3T3 cells had the properties of non-neoplastic, preneoplastic, and neoplastic cells, respectively. The closer the cells were to neoplastic behavior, characterized by continuing growth at high density, the slower they multiplied at lower density. Under the conditions of high population density and low calf serum concentration used in the assay for transformed focus formation, the transformed or neoplastic cells were much more sensitive to killing by methotrexate (MTX) than were non-neoplastic cells in the same culture. This differential sensitivity of neoplastic cells was far more pronounced in molecular, cellular, and developmental biology medium 402 (MCDB 402) than in DMEM. It is associated with the presence in MCDB 402 of folinic acid, known clinically as leucovorin, which is a reduced form of the folic acid present in DMEM. Although leucovorin had been shown to selectively spare normal bone marrow and intestine in animals from the killing effect of MTX on tumor cells, we demonstrate the preferential killing of neoplastic over non-neoplastic cells of the same derivation. Neither neoplastic nor non-neoplastic cells were killed once they had stopped multiplying at their respective saturation densities. The development of the light foci characteristic of the preneoplastic cells was less sensitive to MTX than the formation of the dense foci produced by the fully neoplastic cells. The system should serve as a valuable model to establish basic principles and optimal conditions for selective killing of neoplastic cells by chemotherapeutic drugs.

Nucleolar localization of the Werner syndrome protein in human cells

Werner Syndrome (WS) is a human genetic disorder with many features of premature aging. The gene defective in WS (WRN) has been cloned and encodes a protein homologous to several helicases, including Escherichia coli RecQ, the human Bloom syndrome protein (BLM), and Saccharomyces cerevisiae Sgs1p. To better define the function of WRN protein we have determined its subcellular localization. Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization. Studies of WRN mutant cells lines confirmed the specificity of antibody recognition. No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas. The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins. We have also determined the subcellular localization of the mouse WRN homologue (mWRN). In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus. Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.

Minisatellite instability in severe combined immunodeficiency mouse cells

We have recently found that okadaic acid, which shows strong inhibitory activity on protein serine/threonine phosphatases and tumor-promoting activity in vivo and in vitro, induces minisatellite mutation (MSM). Human tumors and chemically induced counterparts in experimental animals are also sometimes associated with MSM. In the present study, we demonstrated minisatellite (MS) instability in severe combined immunodeficiency (SCID) cells in which the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is impaired. Cells from a SCID fibroblast cell line transformed by simian virus 40 large tumor antigen, SC3VA2, and from an embryonal SCID fibroblast cell line, SC1K, were cloned and propagated to 107 to 108 cells, and then subjected to subcloning. After propagation of each subclone to 107 to 108 cells, DNA samples were digested with HinfI and analyzed by Southern blotting using the Pc-1 MS sequence as a probe. Under low-stringency conditions, about 40 MS bands were detected, with 45% ± 6% and 37% ± 3% of SC3VA2 and SC1K cells, respectively, having MSM. In contrast, cells from the RD13B2 cell line, which was established from SCVA2 by introducing human chromosome 8q fragments, on which DNA-PKcs is known to reside, to complement the SCID phenotype, showed a very low frequency of MSM (3% ± 3%). The high frequencies of MSM in SC3VA2 and SC1K were significant, with no difference between the two. The present study clearly demonstrates that MS instability exists in SCID fibroblasts, suggesting that DNA-PKcs might be involved in the stable maintenance of MS sequences in the genome.

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3′-kinase in NIH 3T3 cells

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3′-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: Facilitated retention in the lung vasculature

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell–cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell–cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.

Protein tyrosine phosphatase PTP1B suppresses p210 bcr-abl-induced transformation of Rat-1 fibroblasts and promotes differentiation of K562 cells

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool—as in speciation—aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

Nuclear structure in normal and Bloom syndrome cells

Bloom syndrome (BS) is a rare cancer-predisposing disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. BLM, the protein altered in BS, is a RecQ DNA helicase. This report shows that BLM is found in the nucleus of normal human cells in the nuclear domain 10 or promyelocytic leukemia nuclear bodies. These structures are punctate depots of proteins disrupted upon viral infection and in certain human malignancies. BLM is found primarily in nuclear domain 10 except during S phase when it colocalizes with the Werner syndrome gene product, WRN, in the nucleolus. BLM colocalizes with a select subset of telomeres in normal cells and with large telomeric clusters seen in simian virus 40-transformed normal fibroblasts. During S phase, BS cells expel micronuclei containing sites of DNA synthesis. BLM is likely to be part of a DNA surveillance mechanism operating during S phase.

“Agrolistic” transformation of plant cells: Integration of T-strands generated in planta

We describe a novel plant transformation technique, termed “agrolistic,” that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as “agrolistic” inserts, as distinguished from “biolistic” inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts.

Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents

Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G2/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes.

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by γ-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to γ-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.

Genetic transformation of HeLa cells by Agrobacterium

Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.