Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.     

Virus-enhanced modulation of cell surface antigens: effect on immune lytic susceptibility.

Monkey kidney cells, upon progressive subculture, became refractory to complement (C)-dependent immune cytolysis by anti-cell serum. Arbovirus infection restored these cells to a state of lytic susceptibility. Similar results were also abtained with antibody-dependent cellular cytotoxicity (ADCC), which is C independent. Antibodies raised against different subcultures varied considerably in lytic efficiency, indicating changing patterns of host cell expression during continous subculture. Taken together with the fact that arbovirus infection festored the lytic efficiency of all antibody preparations to the same degree suggested some form of host cell antigen re-expression as a mechanism. The results obtained in several exploratory experiments indicated that the antigenic re-expression responsible for the restoration of lysis was probably a local or selective rather than a generalized phenomenon. Thus, the amount of host cell surface antigen, measured by the use of mouse anti-cell serum and 125I anti-mouse globulin, was identical in both uninfected lytic susceptible and refractory cells, and decreased in both functional states following infection. Further, the binding of 125I concanavalin A, used to quantify surface glycoproteins, was similar in both lytic refractory and susceptible cells, and in both cases declined folowing virus infection. This result was incompatible with gross "masking" of cell surface antigens by exuberant production of surface coat material in lytic resistant cells. Finally, brief trypsinization of lytic resistant cells yielded an 8-fold increase in immune lysis, a result further consistent with local rather than generalized surface changes. The data were discussed interms of modulation of cell surface antigens affected both by repeated subculture and arboviral infection, and as a possible in vitro correlate of altered self-reactivity.     

Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones

We have analyzed the complexity of a unique tumor-specific transplantation antigen expressed by the murine ultraviolet light-induced fibrosarcoma 1591-RE. This tumor is highly immunogenic and is regularly rejected by normal mice. We have derived a cloned cytolytic T cell line showing a reactivity pattern representative of the cytolytic response of the host rejecting this regressor tumor. Using this T cell line (anti-A), variants of 1591-RE (1591-A-) were selected in vitro that had lost the same antigen as progressor variants of 1591-RE selected by the host in vivo. The in vitro derived variant was then used to generate a second T cell clone (anti-B) that recognized an antigen on the parental tumor that had been retained by the variants derived in vitro. Host-selected progressor variants were also found to have retained this antigen. By selecting for variants in vitro from the parental tumor with the anti-B T cell line, it was shown that the two different antigens (A and B) present on the parental tumor were lost independently of each other. Despite the independence of these two antigens, the host T cell response to the parental regressor tumor was invariably restricted to only the "immunodominant" A antigen.     

Biological attributes of colony-type variants of Candida albicans

Twenty 'commensal' oral or 'pathogenic' vaginal isolates of Candida albicans were examined for colony morphology on malt/yeast-extract and serum-based agar media. Diverse and variable colony morphology was seen on serum agar. In 17 strains, selective subculture of morphologically atypical colonies produced progeny which had reverted to the morphology of the majority of parental colonies. However, in one strain, a highly stable colony variant was isolated which did not revert on subculture. In two further strains, variants were isolated which could be maintained with at least 99% homogeneous colony type by selective colony subculture, but reversion to the parental type or switching to other morphologies occurred at rates of 10(-2) to 10(-4): a rapid switching phenomenon. The relative proportions of mycelial or yeast forms were the main determinants of colony morphology. The variants were biotyped using a selection of biochemical tests. The stable variant differed from its parent in several characters, including rate of production of a proteinase enzyme. The pathogenicity of variants was compared in mice, and both stable and switching variants differed in virulence from their parental strains. Colony-type variation on suitable media is thus a powerful tool in the isolation of mutants or variants of C. albicans which differ from 'isogenic' parents in significant biological properties. Such variants may aid identification and characterization at the molecular level of determinants of, for example, pathogenicity and morphogenesis.     

Morphological description of surface structures on strain B41 of bovine enterotoxigenic Escherichia coli bearing both K99 and F41 antigens

In order to describe morphologically the structures on the cell surface of bovine enterotoxigenic Escherichia coli, variants of reference strain B41 (K99+F41+) either negative for K99 and positive for F41 antigens (variants B41A, B41*C), or phenotypically negative for both antigens (variants B41B1, B41B2, B41*CB), and a transconjugant harbouring the K99 plasmid and expressing the K99 adhesin [transconjugant B41 x H510a:H510(2)] were examined by transmission electron microscopy using negative staining. Several negative staining procedures were tested for strain B41 and variant B41A: direct harvesting of strains into ammonium molybdate (2%, w/v), with bacitracin (50 micrograms ml-1) as wetting agent, gave the best results. Three morphologically distinct structures on the cell surface could be identified in cultures grown on Minca medium. Firstly, thin, filamentous, flexible fibrillar structures, presenting a helical structure and a mean diameter of approximately 3 nm, were recognized as K99 fimbriae, since they were present on strain B41 and on transconjugant H510(2), but not on K99-negative variants nor on the recipient strain H510a. Secondly, coil-like structures with a diameter of about 17-20 nm were observed on strain B41 and on variants B41A and B41*C. These structures appeared to consist of two or more curled filaments (diameter 3 nm) joined to coil on themselves into dense spirals. They were very rare in variants B41B1 and B41B2 and were absent on variant B41*CB and on a transconjugant B41* x B41*CB, which had re-acquired the K99 plasmid and which again exhibited K99 fimbriae. Strains B41 and variant B41A gown at 37 degrees C for 24 h on sheep-blood agar exhibited coiled structures like those seen on Minca medium. In contrast, after growth at 18 degrees C for 48 h (which inhibits the synthesis of F41 antigen), coiled structures were no longer expressed on the cell surface of strain B41 and variants B41A and B41*C. Thus the presence of coiled structures correlated with the expression of F41 antigen in strains and variants, which suggests that F41 had a coiled morphology. Finally, straight fimbriae (diameter 6.5-7 nm) were observed on the cell surface of every strain and variant. Their expression on the cell surface was enhanced by several subcultures in th e static broth, and it was inhibited by subculture on agar, but not by culture at 18 degrees C after serial subcultures in static broth. These facts indicated that the straight fimbriae could be common fimbriae, and excluded their being F41 structures.     

Rat alpha-fetoprotein: in vitro production of four molecular variants by clonal cell lines.

Mass and six clonal cultures derived from Morris hepatoma 7777 by standard tissue culture techniques synthesize and secrete alpha-fetoprotein $\text{in vitro}$. During serial passage, the alpha-fetoprotein which accumulates in the media of these cultures contains two concanavalin A-affinity molecular variants. Each of the concanavalin A-affinity molecular variants shows two electrophoretic variants. Mass and clonal cell populations of hepatoma cells continue to secrete $\text{in vitro}$four molecular variants of rat alpha-fetoprotein known to occur $\text{in vivo}$. These results demonstrate for the first time that individual hepatoma cells have the potential to synthesize four molecular variants of alpha-fetoprotein and that this phenotypic property is maintained during serial subculture $\text{in vitro}$.     

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.     

Periplasmic enzymes in Bdellovibrio bacteriovorus and Bdellovibrio stolpii.

When cells of either Bdellovibrio bacteriovorus 109J or Bdellovibrio stolpii UKi2 were subjected to osmotic shock by treatment with sucrose-EDTA and MgCl2 solutions, only trace amounts of proteins or enzyme activities were released into the shock fluid. In contrast, when nongrowing cells were converted to motile, osmotically stable, peptidoglycan-free spheroplasts by penicillin treatment, numerous proteins were released into the suspending fluid. For both species, this suspending fluid contained substantial levels of 5'-nucleotidase, purine phosphorylase, and deoxyribose-phosphate aldolase. Penicillin treatment also released aminoendopeptidase N from B. bacteriovorus, but not from B. stolpii. Penicillin treatment did not cause release of cytoplasmic enzymes such as malate dehydrogenase. The data indicated that bdellovibrios possess periplasmic enzymes or peripheral enzymes associated with the cell wall complex. During intraperiplasmic bdellovibrio growth, periplasmic and cytoplasmic enzymes of the Escherichia coli substrate cell were not released upon formation of the spherical bdelloplast during bdellovibrio penetration. Most of the E. coli enzymes were retained within the bdelloplast until later in the growth cycle, when they became inactivated or released into the suspending buffer or both.     

Properties of a hybrid between lines sensitive and insensitive to contact inhibition of cell division

Hybrid cell lines have been prepared between 3T3, a line highly sensitive to contact inhibition of division, and cl 1-D, an L cell derivative which is not sensitive. A number of hybrid clones isolated were found to be quite sensitive, indicating that in this respect the 3T3 behavior is the more fully expressed in the hybrid. On serial subculture, the hybrid lines gave rise to variants less sensitive to contact inhibition.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. VII. Dominant expression of variant antigens in somatic cell hybrids

After mutagenesis of mouse mastocytoma P815, it is possible to obtain at high frequency stable tumor cell variants (tum-) that are rejected by syngeneic DBA/2 mice. Most of the variants express one or more new individual antigens specific for each variant, that are detectable in vitro by cytolytic T cells (CTL). Somatic hybrids were prepared either between tum- variants and the original P815 clone, or between different variants. Antigen expression of the hybrids was assessed by using long-term CTL clones that recognize specifically the new antigen present on the variants. Expression of tum- variant antigens behaved as a dominant trait in the hybrids. By submitting the somatic hybrids to selection with CTL clones, it was possible to obtain antigen-loss hybrid variants. The analyses of these antigen-loss variants showed that two variant-specific antigenic determinants associated with one of the variant fusion partners could be lost independently. Like the parental tum- variants, both the (tum+ X tum-) and (tum- X tum-) hybrids failed to form tumors in normal mice but formed tumors in irradiated mice.     

Clonal evolution in human lymphoblast cultures.

We established lymphoblast cultures from normal females heterozygous for electrophoretic variants of glucose-6-phosphate dehydrogenase (G6PD), and the X-linked markers have permitted us to look at evolution of these cell populations in culture. The established cultures were phenotypically heterozygous at onset, having both of the mosaic cell populations resulting from X chromosome inactivation. However, by the tenth subculture, the population of cells no longer reflected the heterozygous genotype in 50% of the cultures, as only a single G6PD isozyme was expressed. The ultimate cell composition seems to be influenced by the initial composition, by the nature of alleles at heterozygous X-linked loci that may provide a growth advantage (or disadvantage), as well as by stochastic events. Our results show that lymphoblast cultures may not reflect the X-linked phenotype of the cells from which they were derived. The fate of such cultures seems to be evolution toward clonal cell populations.     

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores

The effect of germ tube induction on the antigenic variability in C. albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45–47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.Abbreviations GYE glucose-yeast extract agar     

A Simplified Method for the Isolation of Bacteroides nodusus from Ovine Foot-rot and Studies on its Colony Morphology and Serology

A simple method for the isolation of Bacteroides nodosus from foot-rot lesions in sheep is described together with observations on the colony morphology of freshly isolated strains and variant colonies arising on subculture in the laboratory. Serological results from a study of English isolates demonstrate a multiplicity of types based on the K antigens as well as additional antigens common to all strains.     

Production of group- and type-specific antigens during non-permissive infection of dog kidney cells with herpes simplex virus type 2.

Infection of a continuous cell line of dog kidney origin (MDCK) with herpes simplex virus type 2 (HSV-2) resulted in production of little to no new infectious virus. Serial subculture of MDCK cells inoculated with HSV-2 did not permit establishment of carrier cell cultures, as assessed by negative results of plaque assays for infectious virus and radioimmunoassay (RIA) for viral antigens. Group- and type-specific antigens were detected in lysates of non-permissive MDCK cells inoculated with HSV-2 and tested by RIA at 24 hours post-inoculation. Polypeptides produced in permissive (Vero) and non-permissive (MDCK) cell systems were labeled with [¹⁴C]-amino acids and analyzed by polyacrylamide slab gel electrophoresis and autoradiography. During non-permissive infection, two polypeptides of large molecular weight, not found in uninfected MDCK cells, one of which commigrated with a major HSV-2 structural polypeptide, were synthesized and reproducibly detected.     

Antigens on human monocytes identified by monoclonal antibodies

Two antigens (Mo1 and Mo2) present on human peripheral blood monocytes have been defined by lytic IgM monoclonal antibodies. Both antigens are present on greater than 70% of adherent mononuclear cells (predominantly monocytes). Mo1 is expressed by monocytes, granulocytes, and Null cells, but is absent from T and B lymphocytes. Mo2, on the other hand, appears specific for peripheral blood monocytes. Neither antigen is present on Ia-positive B cell lines or on tumor cells from patients with B cell lymphoproliferative malignancies, further excluding the possibility that Mo1 and Mo2 are Ia antigens. Mo1 and Mo2 are, however, present on a significant number of blast cells from patients with monocytic leukemia (both myelomonocytic and pure monocytic variants), but relatively infrequently expressed by cells from patients with acute granulocytic leukemia. These results indicate that Mo1 and Mo2 are unique antigens that may represent distinct stages of late monocyte-granulocyte differentiation.     

H-2 Antigen variants in a cultured heterozygous mouse leukemia cell line

H-2 antigen variants, derived from a heterozygous mouse Friend leukemia cell line by selection with anti-H-2 antisera and complement, were tested for susceptibility to cell-mediated cytolysis, using T-lymphocytes directed against individual H-2 antigens. The cytotoxic cells were generated in the BALB and B10 backgrounds by a combination of in vivo and in vitro immunizations. The phenotypes of the variants inferred from the patterns of resistance or susceptibility to CML were consistent with those presumed from earlier assays using antisera. The one exception was the variant cell line which was H-2D(d-) in assays using antisera, but was H-2D(d+) by CML.     

Generation of tumor-specific transplantation antigens by UV radiation can occur independently of neoplastic transformation

The purpose of this study was to determine whether the UV-associated antigens present on tumors induced in mice by chronic UV irradiation could be induced by in vitro irradiation of cells that were already tumorigenic, or whether their occurrence was associated with the primary neoplastic transformation event. Cells of a nonantigenic, spontaneous fibrosarcoma cell line were exposed to UV radiation in vitro, were cloned, and were tested for antigenic properties. A large number of the clones obtained after UV irradiation of the fibrosarcoma cells were highly antigenic (20 of 39), whereas clones derived from unirradiated cultures were not (0 of 10). The antigenic variants did not induce cross-protection among themselves, but induced only variant-specific immunity in vivo. Several antigenic variants were tested for susceptibility to the action of UV-induced suppressor cells, which seem to recognize a common determinant shared among UV-induced tumors. The variants tested were indeed subject to the activity of the UV-induced suppressor lymphocytes. These results demonstrate that the unique antigenic properties exhibited by UV-induced murine skin cancers are also exhibited by cells exposed to UV radiation in vitro. In addition, they imply that the UV-associated antigens arise as a consequence of exposing cells to UV radiation and that they can occur independently of an initial neoplastic transformation event.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Penetration of Bdellovibrio bacteriovorus into Host Cells

Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.     

Loss of reactivity of HL-A antigens in clonal populations of cultured human fibroblasts during aging in vitro

HL-A antigens from several clones of human fibroblasts lost reactivity with specific antisera during serial subculture in association with a substantial decrease in plating efficiency. Uncloned (mass) parental cultures maintained the same antigen reactivities at comparable stages. The loss of detectable surface antigens during aging of fibroblast clones in vitro is consistent with an immune basis in vivo for age-dependent degenerative and neoplastic disease.