Selection and characterization of cells resistant to diphtheria toxin and pseudomonas exotoxin A: presumptive translational mutants.

Two classes of diphtheria toxin-resistant variants were selected from Chinese hamster ovary (CHO-K1) cells: permeability variants, in which uptake of toxin was impaired, and a new class of cytoplasmic variants, which were cross-resistant to Pseudomonas exotoxin. EF-2 prepared from the cytoplasmic variants was resistant to ADP-ribosylation by either toxin. The evidence presented suggests that these are translational variants possessing a mutationally altered EF-2 gene product. These studies also confirmed that Pseudomonas toxin ADP-ribosylates EF-2 in toxin-sensitive intact cells, as well as in cell-free systems.     

Early- and intermediate-stage variants of simian immunodeficiency virus replicate efficiently in cells lacking CCR5

Primate lentiviruses are thought to use the chemokine receptor CCR5 as the major coreceptor for entry into cells. Here we show that some variants of simian immunodeficiency virus (SIV) replicate efficiently in peripheral blood mononuclear cells (PBMCs) lacking a functional CCR5. There were differences in the replication patterns of sequential variants that evolved during SIVMne infection; the late-stage pathogenic variants were unable to replicate in PBMCs lacking CCR5, whereas the early- and intermediate-stage viruses replicated as well in PBMCs lacking CCR5 as they did in cells with wild-type CCR5. The coreceptor specificities of these sequential variants were compared using indicator cell lines expressing known SIV coreceptors. Among the known SIV coreceptors, there were none that were functional for the early and intermediate variants but not the late-stage variants, suggesting that the coreceptor used for replication in PBMCs may be a coreceptor that has not yet been described. Because some variants replicate with high efficiency in peripheral blood cells using this as yet uncharacterized cellular receptor, this coreceptor may be important for viral entry of some target cell populations in the host.     

An alteration in the phosphorylation of vimentin-type intermediate filaments is associated with mitosis in cultured mammalian cells

Analysis of cultured Chinese hamster ovary (CHO) cells has shown that vimentin exists primarily as two 57,000 dalton isoelectric variants, a nonphosphorylated form and a slightly more acidic phosphorylated form. Similar analyses of CHO cells that were treated with colcemid show the presence of at least two to three additional, more acidic, phosphorylated vimentin isoelectric variants. An increasing 32P-specific activity of these variants suggests that this alteration involves increased phosphorylation. Analysis of 32P-labeled vimentin from colcemid-treated cells indicates that the amount of the additional phosphorylated variants correlates with the accumulation of cells in mitosis. CHO cells enriched in mitotic cells without antimitotic drugs demonstrate the same alteration in the isoelectric focusing pattern of phosphorylated vimentin. When mitotic cells are replated, the amount of additional phosphorylated variants is reduced within 30 min. The data suggest that an alteration in phosphorylated vimentin is temporally related to the alteration in the organization of intermediate filaments in mitotic cells.     

CCR5, GPR15, and CXCR6 are major coreceptors of human immunodeficiency virus type 2 variants isolated from individuals with and without plasma viremia

Human immunodeficiency virus type 2 (HIV-2) is generally considered capable of using a broad range of coreceptors. Since HIV-2 variants from individuals with nonprogressive infection were not studied previously, the possibility that broad coreceptor usage is a property of variants associated with progressive infection could not be excluded. To test this, we determined the coreceptor usage of 43 HIV-2 variants isolated from six long-term-infected individuals with undetectable plasma viremia. Using GHOST indicator cells, we showed for the first time that the only coreceptors efficiently used by low-pathogenic HIV-2 variants are CCR5, GPR15 (BOB), and CXCR6 (BONZO). Surprisingly, control HIV-2 variants (n = 45) isolated from seven viremic individuals also mainly used these three coreceptors, whereas use of CCR1, CCR2b, or CCR3 was rare. Nearly a quarter of all HIV-2 variants tested could infect the parental GHOST cells, which could be partially explained by CXCR4 usage. Use of CXCR4 was observed only for HIV-2 variants from viremic individuals. Thirty-eight variants from aviremic and viremic HIV-2-infected individuals were additionally tested in U87 cells. All except one were capable of infecting the parental U87 cells, often with high efficiency. When virus production in parental cells was regarded as background in the coreceptor-transduced cell lines, the results in U87 cells were largely in agreement with the findings in GHOST cells. HIV-2 isolates from aviremic individuals commonly use as coreceptors CCR5, GPR15, and CXCR6, as well as an unidentified receptor expressed by U87 cells. Broad coreceptor usage, therefore, does not appear to be associated with pathogenicity of HIV-2.     

Membrane skeletal protein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression

The avian analog of mammalian erythrocyte protein 4.1, a structural component of the membrane skeleton, has been identified. It is present at the plasma membranes of avian erythrocytes and lens cells, but has not been found elsewhere in comparable amounts. In chickens, it exists as six variants with molecular masses of 87, 100, 115, 150, 160, and 175 kd. The corresponding polypeptides in turkeys are each about 3 kd smaller, suggesting that all may be encoded by a single gene. These variants have similar solubility properties and nearly identical two-dimensional iodopeptide maps that are similar to those of mammalian protein 4.1, but they are differentially phosphorylated. The three smallest variants are the predominant forms in avian erythrocytes, while the two largest variants predominate in avian lens cells. In contrast, mammalian erythrocytes and lens cells exhibit patterns of variants that are more similar to each other. These results show that only a subset of spectrin-containing cells possess protein 4.1, and that these cells differentially express the variants of protein 4.1 in a manner that may reflect corresponding functional differences.     

YAC-1 MHC class I variants reveal an association between decreased NK sensitivity and increased H-2 expression after interferon treatment or in vivo passage

Two H-2 negative variants of the YAC-1 lymphoma were selected by mutagenization and sequential in vitro selections and compared with wild-type cells for changes in NK sensitivity and H-2 expression after interferon treatment or in vivo passage. The H-2 negative variants and the low H-2 expressor YAC-1 wild-type cells had similar NK sensitivity. However, IFN-beta or recombinant IFN-gamma pretreatments increased the H-2 expression of YAC-1 and protected them from NK lysis, whereas the H-2 variants, which remained H-2 negative, were not protected and often more sensitive to NK lysis. The H-2 variants were similarly susceptible as wild-type cells to three other cellular effects of interferon: protection from virus infection, modulation of Con A capping, and inhibition of cell proliferation. Thus, the only interferon-mediated effect that distinguished the H-2 negative variants from wild-type cells was the inability of the former to increase their H-2 expression and decrease their NK sensitivity. The wild-type YAC-1 line showed increased H-2 expression and decreased NK sensitivity after in vivo passage. In contrast, in vivo passaged H-2 variants showed no reexpression of H-2, and remained NK sensitive. The altered responses to interferon and in vivo passage were specific for loss or down-regulation of H-2, because Thy-1 loss (H-2 positive) YAC-1 variants behaved as the wild-type cells in all respects. This study supports the hypothesis that NK cells may function in vivo to eliminate host cells that fail to express H-2 after interferon stimulation during an immune response; such cells are a potential threat because they may escape recognition by T lymphocytes despite the expression of viral or tumor-associated antigens.     

Somatic variation of H-2Kk expression and structure in a T-cell lymphoma: instability, stabilization, high production and structural mutation

In the heterozygous T lymphoma line LDHB, variants which have lost the expression of individual H-2 class I genes are spontaneously generated in vitro at a frequency of 10(-1)-10(-2). A cell line (HK13) in which the class I gene Kk is stably expressed (frequency of loss variants less than 10(-4) was selected from LDHB cells by fluorescence activated cell sorting. Further selection of HK13 cells for high Kk expression led to the isolation of the HK22 line which expresses twice as much Kk as HK13. From HK13 and HK22 cells, spontaneous structural variants of Kk having lost individual serological determinants of the Kk wild-type molecule, were isolated by fluorescence activated cell sorting. Such variants occur at a frequency of 10(-6)-10(-7) per cell per generation. The analysis of these variants indicates that they carry mutations in the Kk structural gene and that HK13 cells express a single Kk gene which appears to be duplicated in HK22 cells. We did not find evidence for the generation of variants expressing 'alien' class I products in the LDHB cell line. The instability of class I gene expression in LDHB cells and the transition to stable expression may represent steps of T-cell differentiation in the thymus.     

Interleukin-7 in plasma correlates with CD4 T-cell depletion and may be associated with emergence of syncytium-inducing variants in human immunodeficiency virus type 1-positive individuals

Human immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV-positive patients. There was a negative correlation between circulating IL-7 levels and CD4(+) T-cell count in HIV-positive patients (r = -0.621; P < 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/microl than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.     

Viability of the R, S and M variants of Mycobacterium lacticolum under various preservation conditions

The survival rate of Mycobacterium lacticolum and the proportion between its R, S and M variants were studied in the course of 12 months under different conditions of storage (freeze-drying, under vaseline oil, in 0.85% NaCl solution and in distilled water). A high survival rate of the variants was found in cells freeze-dried in a protective medium containing 10% of sucrose +1% of gelatin as well as in a 0.85% solution of NaCl.The survival rate of te variants differed by 2--3 orders of magnitude in cells freeze-dried with sodium glutamate or suspended in distilled water. The proportion between the R, S and M variants in the population noticeable changed after storage under these conditions.     

In vitro selection of cell variants resistant to macrophage and hydrogen peroxide cytotoxic activity in spontaneous transformed cells of the STHE strain]

The new cell variants were selected in vitro from the low-malignant Syrian hamster embryo cells (STHE strain) spontaneously transformed in vitro. Ten cycles of in vitro co-cultivation of the parental STHE cells with the normal Syrian hamsters peritoneal exudate cells (PEC) (resident and LPS-activated PEC) used as a selecting agents (PERr, PECa) were performed. The susceptibilities of thus selected STHE cell variants as well as parental STHE cells to macrophage cytotoxicity and H2O2 dagame were tested with 3H-TdR assays. It has been demonstrated that all five STHE cell variants selected in vitro with PECa, in contrast to the parental STHE cells and three STHE cell variants selected with the use of PECr, were significantly more resistant to H2O2-mediated cytotoxicity. The increased resistance was acquired already after the 1-st cycle of the selection procedure. In contrast, only one STHE cell variant selected with PECr appeared to be semiresistant (demonstrated in 4 out of 6 experiments) to H2O2 damage after 10th cycle of in vitro co-cultivation.     

ATBF1异构体的选择性表达及其抑癌作用的分子机制

ATBF1(AT motif binding factor 1)基因是一个新发现的抑癌基因,其表达产物是目前发现的分子量最大的转录调节因子,它能和甲胎蛋白（alpha fetoprotein, AFP）基因增强子AT富聚区结合,调节AFP的转录.ATBF1基因表达过程中,由于转录本mRNA的选择性剪接,可产生ATBF1-A和ATBF1-B两种异构体,这两种异构体对AFP表达的调节具有相互对抗作用.ATBF1-A是ATBF1基因的主要表达形式,其能抑制癌细胞生长,而ATBF1-B则能促进癌细胞增殖.本文分析ATBF1异构体如何调控AFP表达及其作用的多样性,阐述ATBF1表达下调对肿瘤细胞生长和侵袭产生的影响;探讨ATBF1异构体抑癌作用的可能机制和选择性应用ATBF1异构体治疗肿瘤的科学意义.     

Coordinated changes in gene expression,H1 variant distribution and genome 3D conformation in response to H1 depletion

Up to seven members of the histone H1 family may contribute to chromatin compaction and its regulation in human somatic cells. In breast cancer cells, knock-down of multiple H1 variants deregulates many genes, promotes the appearance of genome-wide accessibility sites and triggers an interferon response via activation of heterochromatic repeats. However, how these changes in the expression profile relate to the re-distribution of H1 variants as well as to genome conformational changes have not been yet studied. Here, we combined ChIP-seq of five endogenous H1 variants with Chromosome Conformation Capture analysis in wild-type and H1.2/H1.4 knock-down T47D cells. The results indicate that H1 variants coexist in the genome in two large groups depending on the local GC content and that their distribution is robust with respect to H1 depletion. Despite the small changes in H1 variants distribution, knock-down of H1 translated into more isolated but de-compacted chromatin structures at the scale of topologically associating domains (TADs). Such changes in TAD structure correlated with a coordinated gene expression response of their resident genes. This is the first report describing simultaneous profiling of five endogenous H1 variants and giving functional evidence of genome topology alterations upon H1 depletion in human cancer cells.     

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.     

Plasmid deoxyribonucleic acid and translucent-to-opaque variation in Mycobacterium intracellulare 103.

After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium intracellulare strain 103, gave rise to variants forming opaque colonies. These opaque variants were more sensitive streptomycin, kanamycin, viomycin, and rifampin than were the wild-type translucent variants. Plasmid deoxyribonucleic acids taken from translucent strain cells and from cells of certain opaque variants were analyzed by agarose gel electrophoresis. Two plasmids of molecular weights of approximately 2 x 10(6) and 50 x 10(6), respectively, were found in the wild-type translucent cells; one of them, the 2 x 10(6)-molecular-weight plasmid, was always missing from deoxyribonucleic acids of the opaque variant cells. The results suggested that translucent colonial appearance and antibiotic resistance of the strain are plasmid-determined functions.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Variants of BALB/c 3T3 cells lacking complex gangliosides retain a fibronectin matrix and spread normally on fibronectin-coated substrates

Evidence has accumulated that di- and trisialogangliosides are involved in the interaction of cells with fibronectin. We have therefore tested the ability of variants of BALB/c 3T3 deficient in such gangliosides to organize a fibronectin matrix and to spread on fibronectin-coated substrates. Whereas BALB/c 3T3 cells contained gangliosides GM3, GM1, and GD1a, direct chemical analysis showed that five out of six variants isolated contained no detectable GD1a. By the overlaying of thin layer chromatograms of cellular gangliosides with 125I-cholera toxin, these variants were also found to lack ganglioside GM1. In contrast, the sialogalactoprotein profile of these cells, analyzed using an 125I-ricin/SDS polyacrylamide gel overlay technique, was similar to that of the parent cell line. All variants organized an extensive fibronectin matrix comparable to that of BALB/c 3T3, as shown using either immunofluorescence or lactoperoxidase-catalyzed iodination. The variants could also spread on fibronectin-coated substrates and adopt a morphology similar to that of BALB/c 3T3 cells, with little or no difference in the concentration of fibronectin required for 50% cell spreading. Cell spreading of the variants was accompanied by the formation of focal contacts and microfilament bundles, in a manner closely resembling that seen with BALB/c 3T3 cells. Treatment of BALB/c 3T3 cells with neuraminidase, which converts much of the cellular GD1a to GM1, did not affect cell spreading on fibronectin. The results clearly demonstrate that complex gangliosides are not essential for retention of a fibronectin matrix or for spreading on fibronectin-coated substrates.     

Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth

At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.     

Haplopappus gracilis cell strains resistant to pyrimidine analogues

Summary Strains of Haplopappus gracilis (Nutt.) Gray cells resistant to 6-azauracil have been isolated from cultures of diploid cells. These strains are also resistant to 8-azaguanine, as is their parent. The variants are 100- to 125-fold more resistant to 6-azauracil than their parent, and they exhibit different spectra of cross resistance to other pyrimidine analogues. The phenotype of each variant is stable in the absence of selection. The majority of cells in cultures of the variants are diploid; all others examined were tetraploid. Initial rates of uptake of uracil are not reduced in the variants. Fluorouracil, to which two variants are resistant, is taken up by one of them as well as by the parent. Responses of the other two to fluorouracil are not correlated with decreased ability to accumulate this analogue.