Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.     

Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.     

Evidence for suppression of cellular growth in vitro and selection against the indigenous mouse X chromosome in A9 cell hybrids after microcell-mediated transfer of an X from other mammalian species

Introduction of a human or Syrian hamster X chromosome (derived from BHK-191-5C cell hybrids) into tumorigenic mouse A9 cells via microcell fusion induced changes in cellular morphology and a retardation of cellular growth. The suppression of growth of the hybrids could be abolished, however, by daily changes of medium containing 20% serum. G-banding analysis showed the absence of a single, cytogenetically identifiable, indigenous X chromosome (marker Z) in two of four hybrid clones after an X chromosome was transferred from either hamster or human cells. All hybrids were tumorigenic when tested in nude mice. Together, these data suggest that the loss of the mouse X chromosome took place probably because of growth inhibitory effects imposed on hybrid cells due to the increase in X chromosome dosage. In addition, our results show a lack of association between the phenotype of cellular growth suppression in vitro and the phenotype of suppression of tumorigenicity in vivo.     

Genetic analysis of tumorigenesis: IV. Chromosome reduction and marker segregation in progeny clones from Chinese hamster cell hybrids.

Hybrid cells produced by the fusion of pairs of cells, one a tumorigenic derivative of CHEF/16 and the other a nontumorigenic derivative of CHEF/18, give rise to clones which are largely tetraploid, but rare reduced hybrids with chromosome counts in the diploid range have been recovered from tumors of hybrid origin. This paper describes the recovery in cell culture of reduced hybrids in the diploid range by selection with 5-bromodeoxyuridine (BrdU) or methylcellulose as well as by growth in culture of cells from excised tumors. All selected subclones were tumorigenic and resistant to BrdU, but they segregated for resistance to 6-thioguanine. Unselected subclones were tetraploid, nontumorigenic, and sensitive to both drugs. These data show that chromosome reassortment as well as extensive chromosome reduction both occur in a small fraction of the population during growth of each hybrid clone.     

Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells.

Both tumorigenic segregant HeLa X human fibroblast hybrids and tumorigenic HeLa (D98/AH-2) cells can be converted to a non-tumorigenic state following introduction of a single copy of a fibroblast t(X;11) chromosome. The translocated chromosome contains approximately 95% of the 11 chromosome and the q26-qter portion of the X chromosome which contains the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Introduction of a human X chromosome has no effect on tumorigenic expression. Suppression of tumorigenicity is relieved by selecting cells which have lost the t(X;11) chromosome by growth in medium containing 6-thioguanine (6-TG). Further, reintroduction of the t(X;11) chromosome into tumorigenic 6TGR cells again suppresses tumorigenicity. Thus, the introduction of a single copy of a human chromosome 11 is sufficient to completely suppress the tumorigenic phenotype of HeLa cells and is suggestive of the presence of tumor-suppressor gene(s) on this chromosome.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

Levels of fos, ets2, and myb proto-oncogene RNAs correlate with segregation of chromosome 11 of normal cells and with suppression of tumorigenicity in human cell hybrids.

The tumorigenicity in nude mice of human carcinoma-derived D98AH2 (D98) cells is suppressed when cell hybrids are made by fusing these cells with normal human diploid cells. Selection for hybrids that have segregated chromosomes results in the recovery of tumorigenic segregants. These segregants have all lost at least one copy of chromosome 11 of the diploid cell parent. Earlier we found that the parental D98 cells had detectable levels of mRNA specific for 13 of 21 proto-oncogenes examined. To determine if transregulation of proto-oncogenes by genes of the normal cell occurs in such hybrids, the steady-state levels of mRNA specific to 22 proto-oncogenes in the parental cells were compared with those of nontumorigenic D98 X human diploid hybrids as well as with those of their tumorigenic segregants and with the cells of the resulting tumors. The only chromosome consistently segregated in the latter was chromosome 11 of the diploid cell. fos and ets2 RNA levels and the amount of fos protein were consistently elevated in the segregants compared with amounts in the original hybrids. An unexpected finding was the inverse relationship for myb RNA that was barely detected in the parental D98 cells but was at least 10-fold elevated in hybrids that did not have segregated chromosomes compared with those that did. These patterns were evident in RNAs prepared from both subconfluent and confluent cell cultures. The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb RNA levels are on chromosome 11 and whether these alterations are causally related to the tumorigenic phenotype of the hybrid remain to be determined.     

Enhanced expression of glucose transporter GLUT3 in tumorigenic HeLa cell hybrids associated with tumor suppressor dysfunction.

Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced Km by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUT3 together with GLUT1 of 70 kDa was also evident in three gamma-ray-induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2-deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N-glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2-deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed.     

Effects of reduced calcium on proliferation and cell viability in tumorigenic and nontumorigenic rat tracheal epithelial cell lines

We have compared the in vitro growth and viability of tumorigenic and nontumorigenic rat tracheal epithelial cell lines over a range of calcium concentrations from 0.003 to 0.85 mM. A greater dependence on calcium for proliferation was seen in the nontumorigenic line as compared to the tumorigenic line at both the colony formation level and in mass cultures. In the latter culture condition, a marked differential effect on cell survival was also demonstrated. These differences in calcium dependence were seen in media containing fetal bovine serum or low concentrations of newborn calf serum and in a serum-free medium developed for these cells. The effect was also independent of the method used for calcium removal i.e., either by chelex treatment or the inclusion of EGTA. Therefore, loss of calcium dependence may be associated with tumorigenicity in rat tracheal epithelial cells offering a selectable marker for neoplastic cells in carcinogen-exposed preneoplastic cell populations.     

Defined, serum-free culture conditions for the GM-micro-clonogenic assay using agar-capillaries

Culture conditions were determined and optimised for the serum-free growth of granulocyte-macrophage (GM) colonies, derived from mouse bone marrow cells, in agar-containing glass capillaries. The standard medium is DMEM/F-12 (1:1) mixture containing per ml: 10 mg BSA, 35 micrograms transferrin, 20 micrograms soybean lipids and 5 micrograms insulin. In contrast to previous attempts by others, GM-colony yield in serum-free medium was found to be equal to that in serum-containing medium (around 25 colonies/capillary) and to correlate satisfactorily with the cell density at seeding.--The role of polyamine oxidases in cell-proliferation experiments could be studied by this microclonogenic assay without interference from any type of serum.     

Phenotypic expression of malignancy in hybrid and cybrid mouse cells

This study has been directed toward the effect of cytoplasmic transfer on the expression of marker properties in hybrid cell systems. Conventional hybrids between two nucleated cells were constructed between tumorigenic and nontumorigenic cells. Cytoplasmic hybrids, or cybrids, were constructed between enucleated chloramphenicol resistant (CAP R) donor cells (cytoplasts) and nucleated recipient cells. Clear-cut evidence for the cytoplasmic transmission of CAP resistance was obtained. Although cytoplasmic transfer had no effect on tumorigenicity or growth in soft agar, preliminary evidence was found that saturation density of the recipient cells could be altered by cytoplasmic addition in cybrids.     

Regulation of growth of cultured hepatic epithelial cells by transferrin

Late-passage cells of a nontumorigenic and anchorage-dependent hepatic epithelial line (WB-F344), which produce insulinlike growth factor II and transforming growth factor beta constitutively, grow in serum-free medium supplemented only with transferrin. In the presence of transferrin, epidermal growth factor further augments population growth, although epidermal growth factor alone is without effect. Insulin, platelet-derived growth factor, and several inorganic iron salts are also ineffective in supporting cell growth in the absence of transferrin; furthermore, these factors do not augment the action of transferrin. The population growth-promoting effect of transferrin occurs at concentrations of 0.5 nM or greater and the maximal effect is reached with a concentration of approximately 6 nM. A lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), can fully mimic the effect of transferrin on the proliferation of WB-F344 cells, but the molar concentration of transferrin. These results suggest that the critical function of transferrin in the proliferation of WB-F344 cells may be in the delivery of iron to the cells. In the absence of transferrin the proliferation of WB-F344 cells is arrested in serum-free medium in the G0/G1 phase, and a period of protein synthesis after the addition of transferrin is necessary before the cells can proceed to S phase and initiate DNA synthesis. Replacement of transferrin causes quiescent WB-F344 cells to cycle parasynchronously. Epidermal growth factor does not alter the length of the latency period prior to S phase but appears to stimulate the uptake of [3H]thymidine subsequently. Transferrin may act as a "competence" and/or "progression" factor, allowing the replication of these epithelial cell in vitro.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

Suppression of tumorigenicity in somatic cell hybrids. IV. Chromosomes of normal human cells associated with suppression of tumorigenicity in hybrids with D98AH2 carcinoma cells

An analysis for cosegregation of chromosomes and tumorigenicity in 52 hybrids of human diploid X D98AH2 human carcinoma-derived cells reveals the consistent presence of four copies of chromosome 11 in all nontumorigenic hybrids (two from each of the parental cells) and a consistent loss of one or two copies of the 11 in all tumor cells derived from tumorigenic hybrids that grow in nude mice. In our earlier study, assays with restriction fragment length polymorphic (RFLP) markers for the cell parent origin of the chromosomes 11 in the hybrids indicated that at least one of the Nos. 11 lost in the tumor cells is from the diploid. Thus both Nos. 11 of the diploid seem to be required for complete and stable suppression of the tumorigenic phenotype. The results of the present study suggest that chromosome 2 may also carry suppressor information, but this causes only partial suppression of the tumorigenic phenotype in the absence of both Nos. 11. On the other hand, when the hybrids contain full complements of the 2 and the 11, suppression is very stable. All other chromosomes except for Nos. 1, 16, 17, 19, and 21 are clearly discordant with suppression. The latter chromosomes are not discordant often enough to allow their exclusion as possible carriers of suppressor information, particularly in the absence of RFLP evaluations. It is clear, however, that if they do carry such information it is not adequate for maintaining a stably suppressed phenotype in the absence of both Nos. 11 of the diploid.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)