Responsibility for truth in research.

For over half a century, cell cultures derived from animals and humans have served researchers in various fields. To this day, cross-contamination of cultures has plagued many researchers, often leading to mistaken results, retractions of results, cover-ups and some out-and-out falsification of data and results following inadvertent use of the wrong cells. Also, during years of examining cultures for purity we learned that many virologists were not too concerned about the specificity of the cultures they used to propagate the particular virus under study as long as the substrate (whatever it might have been) gave optimal virus yield. Polio virus propagates in primate cells, and much research has involved cells from man and various species of primates. In the 1950s a large number of chimpanzees were held in captivity in Africa for extensive studies of the efficacy of polio vaccine in production at the Wistar Institute in Philadelphia and elsewhere. Chimpanzee tissues, particularly kidneys, were thus readily available and could have also provided substrates for polio virus production, since little was known about the purity of substrates and little attention was paid to their specificity at that time.

Biological characteristics and viral susceptibility of a stable dog kidney cell line.

A cell line derived in 1956 from normal dog kidney is described. The cells are epithelial, contact-inhibited, and can be maintained in the same culture vessels for period of more than 2.5 yr. Karyologically, the cells are hypodiploid with a modal number of 72 as opposed to the diploid number of 78. The karyotype indicates male origin of the cells and clonal derivation of extant cultures due to the presence of two marker chromosomes in all metaphases observed. At the 159th passage the dog kidney (DK) cells did not produce tumors in athymic rats. At least 13 viruses of various types produced transmissible cytopathogenic effects in the DK cells, including all of the human influenza viruses investigated.

Specific chromosome loss associated with the expression of tumorigenicity in human cell hybrids.

Four related nontumorigenic and tumorigenic HeLa x fibroblast intraspecific human hybrid cell lines were analyzed to determine whether specific chromosome(s) are associated with the control of tumorigenic expression. The loss of one copy each of both chromosome 11 and chromosome 14 were associated, with a high degree of statistical significance, with the expression of tumorigenicity in two segregants derived from the original nontumorigenic hybrid population. Although the parental origin of the chromosomes could not be established in this study, our preliminary results suggest that complex, genetically determined, regulatory interactions may operate in the control of neoplastic expression.

Characterization of calcitonin- and adrenocorticotropin-producing human cloned cell lines.

We have developed three human cloned cell lines that produce immunoreactive human calcitonin (ihCT) and ACTH (iACTH) as well as exhibit characteristics of cultured neural cells. Clones HMS-41/I, -78/2, and -98/2 were developed from cell lines HeLa AV3, MBA 9812 (bronchogenic carcinoma), and SW 267 (pheochromocytoma), respectively. Karyological analysis of both the parent and the cloned cell lines confirmed the identity of HeLa AV3 and MBA 9812. When grown in serum-free media designed for culturing neural cells, the patterns of production for both ihCT and iACTH varied among the clones. The multiple patterns of hormone production suggest that the mechanisms involved in the biosynthesis, processing, and secretion of these hormones differ among the clones. The clones contain neuron-specific enolase and the putative neurotransmitters beta-alanine and gamma-amino butyric acid, and they respond to cAMP analogs by differentiating, as noted by the extension of neurites (except the HeLa-derived HMS-41/I). The iACTH extracted from cells and synthetic ACTH exhibited equivalent profiles upon isoelectric focusing. The forms of ihCT noted in cell extracts were similar to those observed in extracts of human tumor tissue. Our rabbit antiserum to hCT failed to detect ihCT in those cell extracts prepared for ACTH determination or in extracts of rat pituitaries, but it did detect CT in rat thyroids by both RIA and immunofluorescent procedures. We concluded that our antisera to hCT do not detect the precursor form of ACTH. The availability of these cloned cell lines provides model systems for examining the production of these peptide hormones and the concomitant expression of neural and endocrine characteristics.

Characteristics of membrane and cytosol forms of the mammary tumor glycoprotein molecule MTGP in human breast carcinoma cell cultures and tumors.

The presence, concentration and selected molecular characteristics of the human mammary carcinoma glycoprotein molecule set MTGP, a trace and apparently tumor-specific molecule, were examined in fifteen cell cultures established from mammary carcinomas, tissue from seven mammary carcinomas and control cultures. Both cytosol and membrane-associated forms of MTGP were analyzed, and each was phenotyped by reference to isoelectric point and buoyant density. All cells or tissues of mammary carcinoma origin contained membrane MTGP, whereas cytosol MTGP was undetectable in cell cultures from half of the mammary carcinomas. Neither membrane nor cytosol MTGP were detectable in cells other than mammary carcinomas. Cytosol MTGP could be assigned to three groups by reference to presence, isoelectric point and buoyant density. Membrane MTGP also exhibited heterogeneity between different tumors and could be assigned to three groups by isoelectric point and buoyant density. Each form of MTGP was homogeneous for a given single tumor or cell culture and retained its phenotypic features with passage and cloning. Four general types of MTGP are proposed, through there may be additional fine heterogeneity that cannot be further resolved at this time. These data provide an initial characterization of the membrane form of MTGP and an integrated characterization that is consistent with the concept that tumor-specific antigens may possess both constant regions by reference to antigens recognized by the antisera and variable structure by reference to physicochemical characteristics.

Cross-contamination of cells in culture.

Lists are presented of references to all known publications describing cell properties that serve to characterize (i) known strains of HeLa and purported human cell lines indicated as HeLa contaminants, (ii) strains of human cell lines contaminated with human but non-HeLa cells, and (iii) strains of cells contaminated by cells from one or more other species. Frequencies of cell cross-contaminations are cited and references are presented to relatively simple techniques that could serve to detect such contamination.

Selective killing of human malignant cell lines deficient in methylthioadenosine phosphorylase, a purine metabolic enzyme.

Seven out of 31 (23%) human malignant tumor cell lines had no detectable methylthioadenosine phosphorylase activity (less than 0.001 nmol/min per mg of protein), assayed with 5'-chloroadenosine as substrate. The enzyme-deficient cell lines were derived from five leukemias, one melanoma, and one breast cancer. None of 16 cell lines of nonmalignant origin, derived from lymphocytes, fibroblasts, and epithelial cells, lacked the enzyme (range, 0.156-1.447 nmol/min per mg of protein). As detected by autoradiography, intact enzyme-positive cell lines normal immature bone marrow cells, and four specimens of malignant tumor cells incorporated the adenine moiety of 5'-chloroadenosine into nucleic acids; however, no enzyme-deficient cell lines used 5'-chloroadenosine. When both types of cell lines were cultured in a medium containing 0.4 microM methotrexate, 16 microM uridine, and 16 microM thymidine (or 10 microM azaserine alone), no cells grew. If methylthioadenosine was added to the same medium, only enzyme-positive cells increased in number; most enzyme-deficient cells were dead after 3 days. Thus, human malignant tumor cell lines naturally deficient in methylthioadenosine phosphorylase could be selectively killed when de novo purine synthesis was inhibited and methylthioadenosine was the only exogenous source of purines.

Human embryonic lung cells (HEL-R66) are of monkey origin.

Two cultures of cells designated HEL-R66, presumable of human origin, revealed monkey instead of human cell characteristics, including chromosomes, isoenzyme mobility pattern and species-specific cell membrane antigen.

Contamination of Hodgkin's disease cell cultures.

Several laboratories have recently reported the establishment and characterization of long-term cell lines thought to be related to the neoplastic cell of Hodgkin's disease. Here, Harris et al. discuss evidence that some of these lines are, in fact, not related to Hodgkin's disease but are non-human contaminants.

Tumorigenicity of human malignant lymphoblasts: comparative study with unmanipulated nude mice, antilymphocyte serum-treated nude mice, and X-irradiated nude mice.

The ability of unmanipulated, antilymphocyte serum (ALS)-treated, and X-irradiated nude BALB/c female mice (in their 13th backcross generation) to serve as hosts for 10 human lymphoblastoid cell lines as well as for peripheral blood lymphocytes from healthy humans was compared. The 10 lymphoma lines included 7 previously characterized and reported in the literature. Significant differences with respect to both latency period for tumor appearance and success rate for tumor transplantation were detected among the 3 experimental groups. The unmanipulated mice were poor recipients for lymphoma heterotranplants, and tumors were produced in only two instances. In contrast, 6 tumor lines were successfully transplanted in mice inoculated with ALS, and all 10 lines were successfully transplanted in X-irradiated recipients. Although tumors were readily produced in ALS-treated and X-irradiated mice, no gross or histologic evidence of focal or distant metastases was apparent. Human lymphoblastoid cells were recultured, essentially unchanged, from the heterotransplants from 6 of 7 tumors tested. Although the tumor line HT 1460, originally from a human lymphoma, was successfully transplanted in all 3 groups, only mouse cells were recultured. The use of X-irradiation, rather than ALS, to immunosuppress nude mice offers a more standardized method for heterotransplanting human tumor cell lines and permits ready comparisons between laboratories. Furthermore X-radiation permits transplantation and subsequent study of lymphoblastoid tumors that are otherwise difficult to successfully transplant in nude mice.

Expression of oncodevelopmental gene products by human tumor cells in culture.

Sixty-seven human tumor cell lines and 15 lines derived from normal tissue were examined for the production of the oncodevelopmental markers carcinoembryonic antigen, alpha and beta subunits of chorionic gonadotropin, placental and nonplacental forms of alkaline phosphatase, gamma-glutamyltransferase, cystyl aminopeptidase, and calcitonin. Both intracellular and extracellular levels of these markers were determined at three phases during the growth of each culture. Sixty-eight percent of the cell lines produced elevated levels (greater than or equal to 90th percentile) of at least one marker. Of those, 46% produced elevated levels of one marked only, 29% produced two, 22% produced three, and 4% produced four markers. No cell line produced more than four markers at elevated levels. In most instances, however, the expression of any two particular markers was discordant. For approximately 50% of the possible marker pairs, Spearman rank-ordered correlation analyses showed significant negative correlations, indicating that when one marker was produced at elevated levels by a given cell line, other markers were usually absent ot produced at relatively low levels. In no instance was a significant positive correlation found between two markers. These data indicated that, although most human malignant cells examined produced one or more oncodevelopmental gene markers at elevated levels, no predictable coexpression of any two of the markers was seen.

Characteristics of HeLa strains: permanent vs. variable features.

Characteristic rearranged human chromosome markers have been observed in a variety of HeLa cell sublines and in five suspected HeLa contaminant lines originally thought to be derived from differentiated tissues of different individual patients. The allozyme genetic signatures, representing the composite enzyme phenotype at eight polymorphic loci, of each of the studied contaminant lines were identical to each other and to those of HeLa cells. The probability that each of these lines would have an identical genetic signature (since the frequency of the HeLa genotype is 0.0017) is 4.2 X 10(-15). Differences between cell lines, however, could be detected by isoelectric focussing of the isoenzymes for glucose-6-phosphate dehydrogenase. The cell lines, including CLL 74, the older Chang liver line, failed to express five liver-specific proteins. One protein was detected in a new liver cell culture. Variations in cytogenetic, biochemical, and differentiated functions during continuous cell culture are discussed.

Heterochromatinization, chromatin elimination and haploidization in the parahaploid mite Metaseiulus occidentalis (Nesbitt) (Acarina: Phytoseiidae).

Embryogenic mitoses, mitoses in females and spermatogenesis are described in the predatory mite Metaseiulus occidentalis (Nesbitt). At 22 degrees C, egg development lasts approximately 4 days. Six chromosomes are seen in mitotic metaphases and anaphases of 0--24 h eggs. Toward the end of this period some embryo squashes have patches of cells containing nuclei which are partially heteropycnotic. These patches of cells apparently increase in size with the age of the embryo. In approximately 1/2 of all 24--48 h-old eggs they encompass all or most cells of the embryo. In these embryos metaphases involved 6 chromosomes, anaphases 3. Either prior to, or following metaphase, a pairing of chromosomes appeared to take place to form 3 units which resembled meiotic diplotene chromosomes where there is opening out between homologues. At metaphase, two sets of 3 chromosomes were slightly differentially stained. One, designated the H set, was darker and slightly more contracted than the other, the E set. At anaphase, 3H and 3E chromosomes segregated in a reductional division retaining the differential contraction until telophase. No cytokinesis appeared. The H set appeared to remain contracted while the E set decontracted to assume the appearance of an interphase nucleus. Both of these entities, side-by-side, created the partially heteropycnotic nucleus mentioned above. The H set then appeared to be excluded from the cell. Mitotic meta- and anaphases involving 6 chromosomes were noted in female deutonymphs. Spermatogenesis appeared to encompass an equational division of 3 chromosomes, which the formation of a binucleate spermatid. Two tail structures appeared juxtaposed at the edge of each spermatid and thereafter a separation into two individual sperms occurred.--While mitosis was not studied in known males, we believe that the embryos exhibiting heterochromatinization and elimination of chromosomes in most or all cells were in fact demonstrating parahaploidization.

Establishment of a lymphoblastoid cell line and isolation of an Epstein-Barr-related virus of gorilla origin.

A B-lymphoid cell line was established from a normal gorilla. The cells contained Epstein-Barr virus-related antigens, and herpesvirus particles were demonstrated by electron microscopy. DNA-DNA reassociation kinétics revealed 30 to 40% hybridization to Epstein-Barr virus with 50 genomes per cell. Examination of the viral nuclear antigen with gorilla sera showed this to be a unique isolate termed Herpesvirus gorilla. H. gorilla transformed gibbon B-lymphocytes in vitro.