Unstable Cell Lines Research Articles

Persistent oxidative stress and gene expression changes in radiation-induced genomic instability

In addition to the damage caused by the initial exposure to ionizing radiation, it is now known that ionizing radiation induces delayed DNA damage in cells by a process known as radiation-induced genomic instability. Characterized using many different endpoints including increased mutation, frequency, increased level of reactive oxygen species (ROS), and decreased plating efficiency, radiation-induced genomic instability is best characterized in the GM10115 system by chromosomal instability. While the cellular and molecular mechanism of induced instability is not known, we are investigating the hypothesis that clones exhibiting instability display an altered pattern of gene expression. An 1152 gene microarray chip was used to uncover potential gene expression changes in two sets of two unstable clones in comparison to an irradiated but chromosomally stable clone. One candidate gene, Cu/Zn superoxide dismutase 1 (SOD1), was under-expressed in the unstable clones by an average of 3.66-fold by array analysis. Given the high levels of ROS observed in unstable clones, we performed a detailed analysis of SOD1 in four chromosomally unstable cell lines. Northern and western blotting assays could not confirm the microarray data, showing a decrease in expression of SOD1 in only one of four unstable lines, and increases in average protein level in all four unstable clones relative to control. Average SOD1 activity was also elevated in three of four unstable clones tested. These results indicate that while increased ROS levels are characteristic of many of our clones showing instability, this is not due to under-expression of SOD1 as suggested by DNA microarray analysis.

A549 subclones demonstrate heterogeneity in toxicological sensitivity and antioxidant profile.

In A549 cell culture, significant variability was found in sensitivity to actinomycin D. Using limiting dilution, actinomycin D-susceptible (G4S) and -resistant (D3R) subclones were isolated. G4S cells were also susceptible to protein synthesis inhibitors, a redox cycling quinone, and an electrophile with concomitant activation of caspases 3 and 9. D3R cells were resistant to these agents without caspase activation. Antioxidant profiles revealed that D3R cells had significantly higher glutathione and glutathione reductase activity but markedly lower catalase, glutathione peroxidase, and aldehyde reductase activities than G4S cells. Thus A549 cells contain at least two distinct subpopulations with respect to predisposition to cell death and antioxidant profile. Because sensitivities to agents and the antioxidant profile were inconsistent, mechanisms independent of antioxidants, including the apparent inability to activate caspases in D3R cells, may play an important role. Regardless, the results suggest that antioxidant profiles of asymmetrical cell populations cannot predict sensitivity to oxidants and warn that the use of single subclones is advisable for mechanistic studies using A549 or other unstable cell lines.

Expression of neutralizing recombinant human antibodies against Varicella Zoster virus for use as a potential prophylactic.

Chickenpox is a highly infectious disease that can be life-threatening to certain groups such as the newborn of nonimmune mothers and immunocompromised patients. At present, prophylactic treatment of individuals at risk involves the use of a polyclonal antibody preparation derived from the pooled sera of hyperimmune donors. While this product is effective, there are problems associated with maintaining supply, which depends on the availability of donors, and the variation of potency between batches. An effective human monoclonal preparation would be of value by providing a well-characterized and standardized preparation available on demand. In this study recombinant human anti-varicella zoster virus (VZV) monoclonals were generated from the mRNA of unstable anti-VZV secreting heterohybridoma cell lines, and characterized according to their molecular weight, isoelectric point, glycosylation, binding to C1q, and efficacy at neutralizing VZV in vitro. In one antibody (AEVZ 5.3) the VH region was grafted from the IgG1 parent antibody onto an IgG3 backbone to determine the effect of isotype on neutralization in vitro. Antibodies were expressed from NSO cells at concentrations of 3-24 microg/mL and contained the expected heavy and light chain fragments and N-linked glycan structures. Both AEVZ 5.1 and AVEZ 4 antibodies were IgG1 and recognized the viral coat protein glycoprotein E; both showed complement-independent and complement-enhanced neutralization. Changing the isotype of AEVZ 5.1 from IgG1 to IgG3 (AEVZ 5.3) further enhanced VZV neutralization in the presence of complement, but reduced its neutralization capacity in the absence of complement. Complement enhancement was consistent with our findings that the IgG3 form could bind more molecules of C1q. The results demonstrate the successful use of recombinant methods to generate stable, functional monoclonal antibodies. Modifications of the original antibodies were made with the aim of improving functionality. The resulting cell lines could be used for large-scale production of well-characterized antibodies for therapeutic use.

Impaired repair of DNA double strand breaks in genetically unstable cell lines of malignant melanoma and tumorigenic keratinocytes

Impaired repair of DNA double strand breaks in genetically unstable cell lines of malignant melanoma and tumorigenic keratinocytes

Stable radiobiological features in a genetically unstable glioblastoma cell line

. In a human glioblastoma (grade IV) cell line, A7, the change in chromosome number, growth characteristics, radiosensitivity and DNA-repair capacity were determined during continuous culture over approximately 1300 generations. The median chromosome number fell from 101 to 85 while a remarkable variability was retained. The net population doubling time shortened from 21 to 16 h but no alterations were detected either in radiosensitivity or DNA-repair capacity, as measured by colony and plasmid-reconstitution assays respectively. Reviewing radiosensitivity data from the literature, it is considered that the relative radioresistance of glioblastomas might be inherited from the normal tissue from which they are derived.

Fibroblast growth factor overexpressing breast carcinoma cells as models of angiogenesis and metastasis.

Progression of breast cancer from an estrogen-dependent, slowly growing tumor amenable to tamoxifen treatment to an aggressive, metastatic, estrogen-independent phenotype has been mimicked by the transfection of MCF-7 breast carcinoma cells with fibroblast growth factors 1 or 4. FGF-transfected cells are aggressively tumorigenic in ovariectomized or tamoxifen-treated nude mice, conditions under which the parental cells would not produce tumors. When detection of metastasis was enhanced by lacZ transfection, the FGF-transfected MCF-7 cells were reliably metastatic to lymph nodes and frequently metastatic to lungs, in further contrast to parental cells. An antiangiogenic drug, AGM-1470, given to mice bearing tumors produced by FGF-transfected MCF-7 cells, produced a decrease in tumor size. The decreased tumor size was not as marked as that produced by treatment with pentosan polysulfate, an agent which would abrogate all autocrine or paracrine effects of the transfected FGF. Thus, increased angiogenesis may be a component of the phenotypic change produced by the FGF transfection, but other autocrine or paracrine effects may also be important. Since a clonal FGF-4 and lacZ doubly-transfected cell line, MKL-4, progressively lost expression of the transfected lacZ gene in individual cells, we performed successive rounds of fluorescence-activated cell sorting to select high-expressing cells. High-expressing cell populations thus obtained rapidly lost expression of beta-gal activity in continued culture. High beta-gal expressing clonal cell lines of MKL-4 cells established by either one or two rounds of low-density cloning also lost lacZ expression with continued culture. Southern analysis of DNA from lacZ transfected cell lines showed the transfected sequences to be present and grossly intact in both high and low expressing populations. However, Northern analysis revealed that high-expressing populations of MKL-4 cells contained the most lacZ mRNA, implying that in the unstable MKL-4 cell line, individual cells are down-regulating mRNA levels of lacZ. Stable lacZ expression has been obtained in other FGF-transfected and parental MCF-7 cell lines using the same expression vector. Thus, the MKL-4 cell line is down-regulating mRNA encoding the transfected gene through a mechanism not dependent on the CMV promotor utilized in the expression vector. This evidence suggests that lacZ expression is not a benign modification in certain cells.

BIAcore: a microchip-based system for analyzing the formation of macromolecular complexes.

The formation of macromolecular complexes, for example the complex between an antibody and an antigen, is essential to many biological processes. In order to gain a better insight into the biological processes involved, the formation of complexes is often measured and their interaction kinetics and affinities analyzed. Considerable effort has been expended in developing techniques to measure the formation of complexes, the most promising being electronic microchip-based biosensors. Such analytical systems have come into increasing use recently for the characterization of interactions between biological macromolecules and ligands. One such commercially available system, called BIAcore (Pharmacia Biosensor, Uppsala, Sweden), uses biosensor-based technology and offers a method for rapid analysis of interaction kinetics and affinities. Such systems have the advantage of replacing tedious binding assays utilizing radiolabelled or fluorescent components and potentially unstable cell lines, and can be used where biological activity assays are not available.

Stability of producer hybridoma cell lines after cell sorting: a case study.

Flow cytometry was used in combination with immunofluorescent staining for intracellular and surface-associated antibody contents to identify a significant non-producer cell fraction in a murine hybridoma cell line that had shown a decline in monoclonal antibody productivity with passaging. Viable producer cells stained for surface-associated antibody content were isolated by cell sorting on the basis of surface fluorescence intensity. The fraction of nonproducers was initially reduced from 85% to 20%. Sorting a second time, after these cells were cultivated for 2 weeks, further reduced the fraction of nonproducers to less than 4%. The stability of this purified producer hybridoma cell line during passaging and after a freeze-thaw cycle was investigated. This cell line was found to be highly unstable. A simple batch-growth model simulation was used as follows: (i) to demonstrate that nonproducers appear in hybridoma cell lines after a rare, random mutation event that results in the loss of the heavy-chain gene and/or light-chain gene expression; (ii) to show that a high rate of conversion of producer hybridomas to nonproducer hybridomas cannot entirely explain the population dynamics; (iii) to estimate the rate of conversion of producers to nonproducers to be 8.7 x 10(-5) h-1; (iv) to show that, for this conversion rate, the nonproducer cells' specific growth rates need only be 9% higher than those of the producer cells to dominate the hybridoma culture after only 25 passages; and (v) to predict that producer cells are preferentially lost during cell freezing and thawing procedures. The data suggest that particularly unstable cell lines should be analyzed and purified frequently to prevent overgrowth by nonproducers.

Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy.

The segregation of mutant and wild-type mtDNA was investigated in transformants constructed by transferring human mitochondria from individuals belonging to four pedigrees with the MELAS encephalomyopathy-associated mtDNA mutation (MELAS is mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) into human mtDNA-less (rho 0) cells. Five of 13 clonal cell lines containing mixtures of wild-type and mutant mtDNAs were found to undergo a rapid shift of their genotype toward the pure mutant type. The other 8 cell lines, which included 6 exhibiting nearly homoplasmic mutant mtDNA, on the contrary, maintained a stable genotype. Subcloning experiments and growth rate measurements clearly indicated that an intracellular replicative advantage of mutant mtDNA was mainly responsible for the dramatic shift toward the mutant genotype observed in the unstable cell lines.

A new approach to the generation of human or murine antibody producing hybridomas

A new and very efficient method for the generation of human and murine monoclonal antibodies has been developed. The method is based on clonal expansion of single B cells in the presence of human T cell supernatant and irradiated murine thymoma heiper cells. Subsequently, the clonally expanded B cells are immortalized by electric field mediated cell fusion. The high efficiency of the method permits the processing of small numbers of lymphocytes, e.g., obtained by preselection of specific B cells, small amounts of human donor material and murine PBL or lymph node cells. The method may be an alternative for the EBV-transformation technique used for the generation of human monoclonal antibodies, which immortalized only a subset of B cells and frequently yields poorly growing or unstable cell lines. This report describes the generation of murine anti-HIV and human anti-rubella antibodies combining the clonal expansion of B cells and mini-electrofusion.

Expression of a human monoclonal anti-(rhesus D) Fab fragment in Escherichia coli with the use of bacteriophage λ vectors

A human anti-(rhesus D) antibody (IgG1 lambda) Fab fragment was cloned from an Epstein-Barr-virus-transformed cell line and expressed in Escherichia coli with the use of bacteriophage lambda vectors. The cloned protein is active in binding to human erythrocytes and permits the development of a recombinant reagent for the prevention of haemolytic disease of the newborn. The method offers a rapid and effective means of rescuing human Fabs from potentially unstable cell lines secreting human antibodies.

Parallel development of cadmium resistance and in vitro transformation in cultured Indian muntjac cells.

The development of cadmium resistance in an Indian muntjac cell line has been investigated. The parent cell line is highly sensitive to cadmium ions. Resistance was obtained by continuous growth of cells in low levels of cadmium with stepwise increments. Four cell lines were developed with resistances of between 50- and 200-fold greater than that of the parental line. Early in the development of resistance an unstable cell line displaying extensive chromosomal rearrangement and an elevated sister chromatid exchange frequency was identified. The more stable resistant lines produced from this original cell line have normal karyotypes. Having passed through the initial period of genome rearrangement the resultant cells acquired several characteristics of morphologically transformed cells. It is concluded that long-term exposure to low levels of cadmium can transform cells in vitro concurrently with their acquiring cadmium resistance.

Genetic instability of cancer. Why a metastatic tumor is unstable and a benign tumor is stable.

It is theorized that tumors may be initiated by two methods: by an error affecting one or several oncogenes, or by an error affecting one or several of the genes controlling the stability of the genome. The majority of cells that misexpress an oncogene(s) and that later form a tumor probably form nonevolving benign tumors. A minority of these cells with an activated oncogene(s) (or one of the descendant cells) may also come to misexpress a stability gene(s). A normal cell that misexpresses only a stability gene(s) may form an evolving and genetically unstable cell line that may later misexpress an oncogene(s). A cell or cell line that misexpresses both an oncogene(s) and a stability gene(s) may form a genetically unstable tumor that creates diverse variants, allowing for extensive tumor cell evolution and the acquisition of malignant and metastatic properties.

A sensitive, nondestructive assay for transfected genes.

We have adapted the fibrin overlay assay for plasminogen activators (Jones et al., 1975) into a gene transfer expression assay which has the advantage of being very sensitive and nondestructive. In this assay plasminogen activators convert plasminogen to plasmin, which then degrades fibrin, resulting in clearings in a fibrin overlay. Furthermore, the assay can be used as a signal indicating the efficiency of gene transfer or the loss of introduced genetic elements in unstable cell lines.

Termination of replication of bacterial chromosomes

It is theorized that tumors may be initiated by two methods: by an error affecting one or several oncogenes, or by an error affecting one or several of the genes controlling the stability of the genome. The majority of cells that misexpress an oncogene(s) and that later form a tumor probably form nonevolving benign tumors. A minority of these cells with an activated oncogene(s) (or one of the descendant cells) may also come to misexpress a stability gene(s). A normal cell that misexpresses only a stability gene(s) may form an evolving and genetically unstable cell line that may later misexpress an oncogene(s). A cell or cell line that misexpresses both an oncogene(s) and a stability gene(s) may form a genetically unstable tumor that creates diverse variants, allowing for extensive tumor cell evolution and the acquisition of malignant and metastatic properties.

Amplification of Hypoxanthine-Guanine Phosphoribosyltransferase Genes in Chromosome-Mediated Gene Transferents

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme activities may be elevated in genetically unstable chromosome-mediated gene transferents selected for transfer of the HPRT gene. Increased levels of HPRT polypeptides in unstable mouse L cell gene transferents were demonstrated by two-dimensional gel electrophoresis and immunoprecipitation. No additional polypeptides were found to be overexpressed. HPRT mRNA levels were elevated 10- to 15-fold in the unstable gene transferent GT427C. Southern blot hybridization experiments showed that overexpression of HPRT correlated with a 5- to 15-fold amplification of HPRT gene sequences in two unstable cell lines. Stabilized gene transferents displayed reduced HPRT copy numbers. The amplification of HPRT gene sequences in the unstable transferent GT427C was associated with the presence of multiple minute chromosome fragments. An average of 9.6 fragments was found per metaphase, but the variation was considerable, ranging from 0 to 53. We conclude that genomic DNA sequences may be amplified in unstable chromosome-mediated gene transferents and that such amplification may be associated with the occurrence of multiple chromosomal fragments.

Amplification of hypoxanthine-guanine phosphoribosyltransferase genes in chromosome-mediated gene transferents.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme activities may be elevated in genetically unstable chromosome-mediated gene transferents selected for transfer of the HPRT gene. Increased levels of HPRT polypeptides in unstable mouse L cell gene transferents were demonstrated by two-dimensional gel electrophoresis and immunoprecipitation. No additional polypeptides were found to be overexpressed. HPRT mRNA levels were elevated 10- to 15-fold in the unstable gene transferent GT427C. Southern blot hybridization experiments showed that overexpression of HPRT correlated with a 5- to 15-fold amplification of HPRT gene sequences in two unstable cell lines. Stabilized gene transferents displayed reduced HPRT copy numbers. The amplification of HPRT gene sequences in the unstable transferent GT427C was associated with the presence of multiple minute chromosome fragments. An average of 9.6 fragments was found per metaphase, but the variation was considerable, ranging from 0 to 53. We conclude that genomic DNA sequences may be amplified in unstable chromosome-mediated gene transferents and that such amplification may be associated with the occurrence of multiple chromosomal fragments.

Phenotype stabilisation and integration of transferred material in chromosome-mediated gene transfer.

The human genes for thymidine kinase, galactokinase and procollagen type I, located on chromosome 17, have been transferred to cultured mouse cells. In unstable cell lines the transferred genetic material is shown to exist independently, whereas in stable cell lines the transferred material has become associated with chromosomes of the recipient cell. The size of the transferred genetic material exceeds 1% of the human haploid genome in some of the transformed cell lines. In addition, the data indicate a gene order of: centromere–galactokinase–(thymidine kinase, procollagen type I), on the long arm of human chromosome 17.