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Chinese hamster somatic cell hybrids between diploid anchorage-independent CHEF/204Bu50 cells and diploid anchorage dependent CHEF/205-30 cells are anchorage dependent but can segregate subclones at low frequency which reexpress anchorage independence. Thus, anchorage independence, like other characteristics of the transformed phenotype, is suppressed in these hybrids. Anchorage-independent subclones were recovered from the anchorage-dependent hybrids under conditions which favored the retention of most chromosomes. Karyotype analysis of suppressed hybrids and their anchorage-independent subclones showed that segregation of anchorage dependence was correlated with the loss of one copy of chromosome 1 in CHEF Chinese hamster hybrids. Thus, suppression of anchorage independence has a chromosomal basis. Several genetic models are considered for the origin of anchorage-independent subclones from suppressed Chinese hamster hybrids.

Chinese hamster embryo fibroblast cell line CHEF/18 is stably diploid, anchorage-dependent, has a high serum requirement, and does not form tumors in nude mice. The chromosome constitutions of spontaneous and chemically induced anchorage-independent and/or low-serum CHEF/18 mutants and tumors produced in nude mice by some of these mutants are compared. We find a correlation between diploidy and nontumorigenicity among the anchorage-independent mutants but not in the low-serum mutants. One of the four spontaneous and six of the 15 chemically induced anchorage mutants have remained diploid. The remaining 12 mutants are pseudodiploid or aneuploid, and seven of them contain changes in chromosome 1, either a translocation or a deletion involving breakage at the same position (1q11–12). Each of the tumors induced by six mutants has a unique pattern of rearrangements;however five of the six have changes involving chromosome 3. This chromosome was also frequently rearranged in tumorderived cells previously investigated.

In many hybrid cell systems fusion of normal cells to tumorigenic cells suppresses the transformed phenotype. We have now examined the expression of anchorage independence, one characteristic of the transformed phenotype, in hybrids between different anchorage-independent Chinese hamster fibroblasts. Examination of many different crosses was facilitated by a rapid screening procedure. Instead of isolating individual hybrid clones, the fraction of hybrid cells that were anchorage independent was estimated directly after fusion and compared to the total hybrid recovery. Seven different anchorage-independent cell lines were crossed to derivatives of the previously characterized CHEF/16 tumorigenic cell line. Two of these cell lines gave anchorage-independent hybrids, while the 5 others gave anchorage-dependent hybrids. These results support previous observations with interspecies hybrids that the transformed phenotype can be suppressed in hybrids between transformed cells. We interpret these results as showing that there are at least two alternative genomic changes by which Chinese hamster fibroblasts can become anchorage independent.

The chromosome constitution of CHEF/16 clones recovered from methylcellulose and of uncloned, tumor-derived CHEF/16 populations are compared. Nine of 11 clones recovered from methycellulose were initiated by diploid cells. Moreover, chromosomally diploid cells were still present in most CHEF/16 clones even after growth in anchorage-independent conditions. In contrast, none of the CHEF/16 cells recovered from tumors were diploid. Nonrandom chromosome changes were observed, but no specific chromosome alterations were consistently found in tumor-derived CHEF cells. Although CHEF/16 cells are uniformly tumorigenic innude mice, each of 10 uncloned tumor-derived populations from inocula of 102, 104, and 106 CHEF/16 cells consisted of only 1–3 stemlines. Our results show that diploid CHEF/16 cells are premalignant and undergo karyotypic changes leading to successful and usually clonal establishment of tumors innude mice.

The chromosomal constitution of four established diploid Chinese hamster embryo fibroblast (CHEF) cell lines is described. CHEF/18 exhibits anchorage-dependent growth and is not tumorigenic in nude mice. CHEF/16 has a high plating efficiency in methylcellulose and is highly tumorigenic in nude mice. Both CHEF/18 and CHEF/16 have a normal Giemsa banding pattern and constitutive heterochromatin distribution characteristic of normal diploid Chinese hamster cells and exhibit relatively little chromosomal variation within their cell populations. These results suggest that the nuclear changes responsible for tumorigenicity of CHEF/16 involve alterations below the level detectable by Giemsa banding analysis. Chromosome rearrangements were detected in two other CHEF cell lines, CHEF/205-30, a diploid, thioguanine-resistant derivative of CHEF/18, and CHEF/204-Bu 50, a diploid, 5-bromodeoxyuridine-resistant derivative of CHEF/16, which are being used as genetic markers in intraspecific somatic cell hybrids.

We report here that glucose, as a carbon source, and pyruvate are required for the phenotypic expression of cytoplasmically transmitted chlor-amphenicol-resistance (CAP-R) mutations, recovery of CAP-R mutants, and continuous growth in the presence of oligomycin or antimycin. We assume that glucose supplies additional energy when mitochondrial respiration is diminished and that pyruvate provides intermediates when the Krebs cycle is inhibited. Thus, the requirement for pyruvate is fully satisfied by an exogenous source of purines, and partially by a-ketoglutarate or a pyrimidine source. Based upon these findings, we have obtained two types of mutations affecting mitochondrial function—oligomycin resistance and pyruvate-independent expression of chloramphenicol resistance. Both are cytoplasmically transmitted and provide new markers for a genetic analysis of mitochondrial biogenesis.

Two new fibroblast cell lines from a male Chinese hamster embryo are described: one tumorigenic in nude mice (CHEF 16) and the other not (CHEF 18-1). Both lines have a stable diploid mode and mean of 22 with 10 pairs of homologous autosomes. The cell lines differ unambiguously in cloning ability in methylcellulose, colony morphology, and tumorigenicity; the expression of these traits was examined in a set of 18-1 × 16-2 hybrid clones. The results show initial suppression of tumorigenicity and anchorage independence, followed by growth in the nude mouse of subsets of cells with chromosomes reduced to the diploid range. Further studies are in progress to establish whether ability to grow in methylcellulose and in the nude mouse segregate coordinately at the cellular level, and whether this segregation has an identifiable chromosomal basis.

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.

Somatic mouse cells hybrids formed by fusion of nontumorigenic 3T3 and closely related SV40-transformed SVT2 cells were analyzed in a study designed to probe the genetic basis of the multiple phenotypic changes induced by SV40 transformation. These hybrids showed noncoordinate expression of the transformation phenotype. Although they cloned at high efficiency in medium with low serum and expressed the SV40 T-antigen of the SVT2 parent, hybrid cells grew poorly without anchorage and exhibited a cell and colony morphology intermediate between that of the parents. Tumorigenicity was assayed quantitatively by subcutaneous coinjection into athymic nude mice of serial dilutions of 102 to 105 hybrid cells with 107 lethally irradiated 3T3 cells. The results showed that 100–1000 times more hybrid cells had to be injected for tumor formation than were required with SVT2. These and other observations show that most 3T3/SVT2 hybrid cells are not tumorigenic but that each population contains a rare subset of tumorigenic cells.

Polyethylene glycol (PEG) can be used to promote the fusion of enucleated cytoplasts from chloramphenicol (CAP)-resistant mouse cells with intact cells, resulting in the formation of viable cybrids. The techniques are simple and highly efficient, yielding up to one viable cybrid per 20 intact cells fused. It also seems that PEG can be used to induce cybrid formation without the necessity of prior enucleation of the CAP-resistant cells.