Biomedical subjects
T R Chen
Publications and source records attributed to T R Chen.
Mycoplasma infection of cultured cells.
Mycoplasma contamination is tough to detect and even more difficult to eradicate. It is best to start over fresh from clean cell stocks, but several elimination options are available.
SK-UT-1B, a human tumorigenic diploid cell line.
SK-UT-1B (ATCC HTB 115) produced tumors 2 weeks after subcutaneous injection of 10 million cells into athymic nude mice. Karyotypic analysis at the 500-600 subband level, however, did not reveal any noticeable aberrations. This cell line therefore may represent the first example of experimentally proven human tumorigenic cells of solid tumor origin that retains the diploid karyotype in vitro indefinitely.
Re-evaluation of HeLa, HeLa S3, and HEp-2 karyotypes.
In contrast to earlier reports, this study on HeLa, HeLa S3, and HEp-2 revealed that karyotypes of each cell line are characterized by a consistent marker chromosome composition and a constant number of copies for both normal and marker chromosomes. Based on these chromosome fingerprints and an analysis of 50 metaphases, the modal karyotype of each cell line was defined. Each modal karyotype had the composite content of the previously reported karyotypes of the same cell line, and, generally, the former had the same or a higher number of copies per chromosome than the latter. This modal karyotype can be used as a standard to identify and further individualize both the cell line itself and a subline within that cell line. We have also found that many cells within each cell line have the same karyotype. Portions of numerical data are compiled in a chart format by which the extent of chromosome differences between cultures can readily be compared. Also discussed in brief are characteristic chromosome changes that may help distinguish clonally derived cell lines from lines derived by cross-contamination.
Characteristics of cell lines established from human colorectal carcinoma.
We have characterized 14 human colorectal carcinoma cell lines established from primary and metastatic sites by us during the years 1982 to 1985. Five lines were established in fully defined ACL-4 medium and 9 in serum supplemented R10 medium. However, after establishment, cultures could be grown interchangeably in either medium. The lines grew as floating cell aggregates in ACL-4 medium, while most demonstrated substrate adherence in R10 medium. The lines had relatively long doubling times and low cloning efficiencies. Twelve were tumorigenic in athymic nude mice when injected s.c., and two grew i.p. as well. Based on culture, xenograft, and ultrastructural morphologies, the 14 lines could be subtyped as follows: 4 were well differentiated; 5 were moderately differentiated; 4 were poorly differentiated; and 1 was a mucinous carcinoma. Membrane associated antigens characteristic for gastrointestinal cells (carcinoembryonic antigen, CA 19-9, and TAG-72 antigens) were expressed by 50-71% of the lines. Lines expressing carcinoembryonic antigen and CA 19-9 actively secreted these antigens into the supernatant fluids while TAG-72 antigen was not secreted. Surprisingly, 5 of 7 of the original tumor samples tested and 13 of 14 cultured lines expressed L-dopa decarboxylase activity, which is a characteristic enzyme marker of neuroendocrine cells and tumors. In addition, one poorly differentiated cell line contained dense core granules, characteristic of endocrine secretion. Preliminary cytogenetic analyses indicated that 9 of 11 lines examined contained double minute chromosomes. In addition, 3 of the 9 lines with double minutes also had homogeneously staining regions. These findings indicate a high incidence of amplification of one or more as yet unidentified genes.
Cytogenetics of somatic cell hybrids. II. Modal karyotypes and mosaicism in mouse mutant cell lines: LM(tk-), A9, and Rag.
Modal karyotypes of mouse mutant cell lines, LM(tk-), A9, and Rag, which are frequently used as a donor line for somatic cell hybridization, are described and compared with karyotypes previously reported by other laboratories. Results are summarized as follows. Many cells (18%-38%) in each line had the same chromosome composition as the modal karyotype. Chromosome differences from the modal karyotype in some cells mainly involved one or a few chromosomes only. These differences are mostly caused by structural changes from well documented mechanisms or simple numerical changes including artifacts arising from the rupture of cells. Karyotypes of coexisting subpopulations within the same cell line can be clearly identified. Assessment of karyologic properties of a heteroploid cell population may vary considerably depending on whether it is estimated on a subpopulation or the total population as a unit. Modal karyotypes of these cell lines contained most marker chromosomes of, and a similar chromosome composition to those previously reported by other laboratories. These cell lines exhibit karyotypic constancy; thus, we believe that chromosome changes associated with clonal growth of somatic cell hybrids can be identified without ambiguity.
WiDr is a derivative of another colon adenocarcinoma cell line, HT-29.
This paper documents chromosomal and isozymic evidence indicating that WiDr is a derivative of another colon adenocarcinoma cell line, HT-29, which was established in 1964. The phenotypes for seven isozymes studied were identical for all HT-29 and WiDr cell lines. The karyotypes were similar between four HT-29 cultures separated by more than 100 passages and two WiDr cultures that were studied independently in 1979 and 1985. The isozymic and karyotypic data are consistent with that described for the cell line WiDr, originally reported in 1979. Detailed karyotypic comparisons further show that cultures of WiDr possess all nine chromosomal markers found in the initial HT-29 (passage 19) cultures. However, both the higher passaged HT-29 and WiDr differ from the initial HT-29 (passage 19) because it has an additional five to eight constitutive marker chromosomes. An increase or decrease of one copy in several normal chromosomes is also noticed with more normal chromosome gains than losses. The study provides another example demonstrating the remarkable karyotypic stability within a heteroploid cell line with continued passage. It also reemphasizes the critical need for detailed karyotypic and isoenzymic studies on all cell lines in common use.
Evidence for a multistep pathogenesis in the generation of tumorigenic cell lines from hemopoietic colonies exposed to Abelson virus in vitro.
The present studies were undertaken to investigate the ability of Abelson murine leukemia virus (A-MuLV) to transform cells derived in vitro from pluripotent hemopoietic progenitor cells of high proliferative potential. We now report that continuously growing, autonomous cell lines could be obtained from a high proportion of individually infected multilineage colonies generated in assays of spleen cells from normal adult mice if the infected cells were cocultivated for the first two to three months with irradiated NIH-3T3 cells. No lines were obtained if the 3T3 cell feeders were not initially present. Similar results were obtained when the cells exposed to virus were from multilineage colonies originating from isolated single cells obtained by replating small blast colonies. Characterization of the transformants and a number of derivative cloned sublines revealed the consistent presence of a mast cell phenotype, with some suggestion of macrophage differentiation in a few cases. All cell lines tested produced virus, showed a variable pattern of A-MuLV integration, and gave rise directly to tumors when injected subcutaneously, as shown by both Southern analysis and cytogenetic studies. The early absolute but transient dependence of these A-MuLV mast cell transformants on a fibroblast feeder suggests a multistep process in their evolution, in which the acquisition of autonomy from factors of mesenchymal cell origin may play an important role.
Modal karyotype of human leukemia cell line, K562 (ATCC CCL 243).
The chromosome composition of the human leukemic cell line, K562, deposited at the American Type Culture Collection (ATCC) (CCL 243), was studied and further compared with those reported by other laboratories. In CCL 243, the modal karyotype had 55 normal and 14 constitutive marker chromosomes and occurred in nearly 50% of the cultured cell population. Other variant karyotypes were observed either at a low frequency in coexisting minor cell types or only once in all other analyzed metaphases. Normal chromosome #9 and the chronic myeloid leukemia (CML) specific Ph/t(9;22) were absent. It appears that karyotypes of most K562 cultures distributed world-wide are similar to each other, with the stable modal chromosome number, and therefore, the modal karyotype presented here should be useful in karyotypic identification of a cell line.
The chinese hamster Don cell line differs from normal diploidy by one chromosome band.
G-banded and C-banded prometaphase and metaphase karyotypes of the Chinese hamster Don cell line from three different laboratories were compared. All the cell lines were pseudodiploid. Each consisted of two distinct cell populations designated S1 and S2. The chromosome complements of each of these populations differed from the diploid by only one additional chromosome band on a subterminal 1p in S1 and on a median 1q in S2. The sites of these extra bands were neither constricted nor heterochromatic. All other different karyotypes, including those from seven distinct subpopulations, had basic patterns of chromosome complements that were best represented by S1 or S2, respectively the major or minor cell type of the cell line. Moreover, nearly 80% of the metaphases analyzed had a modal chromosome number of 22, and about 60% had the same chromosome composition of a cell type or subtype. Our data also suggest that Don cells were probably pseudodiploid by 1964 or before.
Intercellular karyotypic similarity in near-diploid cell lines of human tumor origins.
Intercellular karyotypic compositions were studied in seven near-diploid cultured cell populations (including six cell lines) of human colorectal and fibrosarcoma origins. Within each population, 50-88% of cells had the same cell-line-specific karyotype. In five of these seven populations, coexisting minor cell types could readily be identified, implicating the frequent occurrence of mosaicism in transformed cell lines. Nearly 90% of these cell types showed a single chromosome loss or gain from the modal cell karyotype. Parameters that may assist in the evaluation of karyotypic diversity of a transformed cell line are discussed briefly.
Chromosome changes in 6-TG-resistant mutant strains derived from a karyotypically stable human line, C32.
Karyotypes of the human C32r16 line and its HPRT-mutant derivatives were compared. All three HPRT-mutant strains (TG-A, TG-E1, and TG-E2) studied were hypotetraploid and arose by genomic duplication. TG-A and TG-E had distinct karyotypes, reflecting their independent clonal origins. Both TG-E1 and TG-E2 were similar karyotypically, except that TG-E2 differed from TG-E1 by the presence of two new sets of balanced translocations and a significantly higher number of new markers, viz., 24 new markers in 14 TG-E2 cells as compared to 2 new markers in 13 TG-E1 cells. Comparison of the hypothetical 2s karyotype of C32r16 with the modal karyotypes of the mutants revealed (1) that chromosome changes occurred more frequently among marker chromosomes (57.1%) than among normal chromosomes (18.1%) and also more frequently among tetrasomic (28.6%) than among disomic (3.3%) normal chromosomes, and (2) that one member of a group of paired marker chromosomes tended to be eliminated very frequently, whereas disomic normal chromosomes retained their disomic condition. Chromosome 7 was involved in the formation of new marker chromosomes twice as much as any other chromosome, and even increased to pentasomy in TG-E1. Chromosome changes associated with 6-thioguanine treatment are briefly discussed.
Karyotype consistency in human colorectal carcinoma cell lines established in vitro.
Karyotypes of nine human colorectal cell lines deposited with the ATCC were studied by trypsin-Giemsa banding. CCL 229,230,231 and 235 (Modal chromosome number, Sm, was 49, 68, 47, and 40, respectively) belong in the stable type that is characterized by karyotypes consisting mostly of normal chromosomes and stable markers. CCL 228, 234, and 238 (Sm=55,79, and 70 respectively) belong in the unstable type that has karyotypes consisting of numerous markers in addition to normal chromosomes and stable markers. The remaining intermediate type (CCL 233 and 237, Sm = 60 and 64, respectively) has karyotypic characteristics between the above two types usually with two or less unstable markers per cell. The stable markers (together with normal chromosomes) are constitutive components of a cell genome and are common to most cells within the same cultured population. Unstable markers, which generally constitute only a small portion of the total chromosome complement are the likely cause of karyotypic variations between cells and often are produced by balanced inter- or intrachromosome changes, or both. Consequently, total chromosome length per cell genome is remarkably consistent within a cell population, and karyotypes between cells, such as from four stable lines, are profoundly stable and mostly identical. Chromosome deletions and interhomologue exchanges (including isochromosomes) had the highest incidences among both stable and unstable markers. The complex markers occurred relatively infrequently. There were neither common markers nor unique chromosome breakages common to all of these established cell lines. However, chromosomes No. 7 and 1 had the highest incidence (15 and 12, respectively) of structural modifications resulting in the formation of stable markers (82 total exchanges in nine cell lines), and chromosomes No. 7 and 2 were involved at high incidence (21 and 15, respectively) in the formation of both stable and unstable markers (181 total exchanges). Moreover, No. 7 is overrepresented in eight of nine lines. The significance of chromosome changes involving No. 7 in this as well as other tumor pathotypes is briefly discussed.
Polymorphic variants in human chromosome 15.
We found eight polymorphic variants in human chromosome 15 using Q, C, Q-C and Ag-NOR staining methods. These variants included brightly or dully fluorescent pericentric segments and satellites, giant satellites, increased amounts of short arm hetrochromatin (ph+) and darkly (C band-positive) or lightly (C band-negative) Giemsa-stained pericentric Q-negative segments. These staining properties indicated that the entire short arm of 15 contained at least four distinct chromatin segments: Q-negative centromeric heterochromatin, a Q-variable distal segment, a Q-negative satellite stalk, and Q-variable satellites, in that order, from proximal to distal ends. The BrdU-Hoechst 33258-stained R bands (RBH) and high resolution G subbands were also studied for karyologic characterization of chromosome 15. Most of these variants were reported also in 13, but insufficiently documented in other D and G chromosomes. Together with polymorpic pericentric fluorochromes seen in 3 and 4, Yq, and nonpathogenic t(D;Yq), the pattern of these variants can be used as karyologic fingerprints for identification of each individual and his or her cell explants both in vivo and in vitro.
Karyotype analysis of a human mammary sarcoma explant in vitro.
Karyotype of a fibroblastic SH-1 cell line derived from a human mammary sarcoma had 86 (80-92) chromosomes including 6 (4-7) markers, and a various number of double minutes. There were generally even numbers (e.g., 2, 4, etc.) of chromosomes in 17 intact and 3 common marker chromosomes. The t(3;14) and del(14), which had 2 each per s cell genome, were probably formed by the balanced translocation between nos. 3 and 14. Three other markers were single and contained a partial or entire no. 1. The presence of these latter markers was often accompanied by the decrease in the number of the intact no. 1, indicating that no. 1 was the only chromosome actively engaged in rearrangements during this stage of karyotypic diversification. Of the no. 2 and 7q that were over-represented on the per cell level, the 7q had a similar observation from many other solid tumors found in this and other laboratories. In contrast, none of the under-represented chromosomes had the concordant changes consistent to other tumors, although the frequent involvement of no. 9 in the chromosome rearrangements was seen in some human heteroploidies.
Frequencies of sister chromatid exchanges in heteroploid cell lines of human melanoma origin.
Sister chromatid exchange(s) (SCE) per cell and chromosome in five heteroploid cell lines and sublines of human melanoma origin were distinctly more numerous than those in the normal diploid control. The SCE per unit chromosome length was higher in stable heteroploid C32 (0.041-0.047) and C9 (0.056) than in normal diploid control. The SCE per unit chromosome length was higher in stable heteroploid C32 (0.041-0.047) and C9 (0.056) than in normal diploid control C182 (0.036), which suggested that the longer the total chromosome length of the cell genome, the higher the SCE per cell as well as per unit chromosome length. These malignant heteroploid lines had karyotypes ranging from hypodiploidy to hypotriploidy and were extremely heterogeneous in one cell line but unusually homogenous and stable in others. However, none of these properties could be correlated to the increase in SCE frequency. As compared to C32-r7, the subline C32-RO had more SCE per cell, chromosome, and unit chromosome length for the genome as well as for 9 of 11 individual chromosomes and chromosome groups. In contrast, the C32-RO specific ring marker chromosome had only 40% of the SCE of its nonring homologue in C32-r7 cells. Interestingly, C32-RO and its ring marker were less stable than C32-r7 and the nonring marker during in vitro growth. There was no noticeable difference between C32-r7 and its derivative C32-r7-nu-1, which implied that transplantation into nude mice did not alter the expression of SCE in this subline.
Cytogenetics of somatic cell hybrids. I. Progression of stemlines in continuous uncloned cultures of man-mouse cell hybrids.
Karyotypes of hybrid cells were studied in continuous uncloned cultures by Q- and C-bandings. Cultures were initiated by virus-mediated or spontaneous cell fusions from normal human diploid fibroblasts and mouse heteroploid RAG cells. Heterokaryons containing complete genomes of both parental cells randomly lost chromosomes from both species. The majority of cells in early growth stages, however, still possessed a nearly complete human genome. The rate of human chromosome loss in subsequent growth periods was not uniform, being gradual in some and rapid in others. The initially predominant 2n human-1s mouse (1h:1m) type was soon replaced by a less frequent 2n human-2s mouse (1h:2m) type. Over an increased period of time in mass culture, the number of stemlines decreased. One stemline, often a (1h:2m) type with a greatly reduced human complement, outgrew the others and occupied the entire culture. Therefore, the usual process of clonal isolation may confer a negative selection bias against cell hybrids retaining a large number of human chromosomes. Hybrid stemlines with stable karyotypes were established in the present HAT-agar selection system before 36 days after fusion had elapsed.
Evolution in vitro of stemlines with minimal karyotypic deviations in a human heteroploid cell line.
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