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At least 19 recordsLinked to original sources
PubMed2003

ING1b decreases cell proliferation through p53-dependent and -independent mechanisms.

ING1b can stimulate cell cycle arrest, repair, senescence, and apoptosis. The actions of ING1b are attributed to its activation of the tumor suppressor p53. Here we investigate the more subtle effects of ING1b on the cell cycle and DNA damage responses in the absence of p53. To this end, we have generated isogenic cell lines that expressed ING1b and p53 either individually or in combination under the control of inducible promoters. A five- to 10-fold induction of ING1b over the endogenous protein in a p53-null H1299 background slightly impairs proliferation by increasing the doubling time by approximately 10%. Significantly, ectopic expression of ING1b enhanced the G(2)/M DNA damage checkpoint induced by adriamycin. We demonstrated that the DNA damage-induced cell death mediated by the cooperation between ING1b and p53 was more prominent than by the individual proteins alone. In adriamycin-treated cells, p53 was stabilized and induced the expression of p21(CIP1/WAF1), but the expression of ING1b was not affected. The exact targets of ING1b in the p53-null background are not known, but we demonstrated that the transcriptional activities of other members of the p53 family, p63alpha and p73alpha, could be activated by ING1b. These data indicate that ING1 has a subtle antiproliferative effect even in the absence of p53, and ING1b enhances the DNA damage responses through p53-dependent and -independent mechanisms.

Cell Cycle Proteins↗
PubMed2005

Hyperphosphorylation of the BARD1 tumor suppressor in mitotic cells.

Although the BRCA1 tumor suppressor has been implicated in a number of cellular processes, it plays an especially important role in the DNA damage response as a regulator of cell cycle checkpoints and DNA repair pathways. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein, and many of its biological functions are mediated by the BRCA1-BARD1 complex. Here, we show that BARD1 is phosphorylated in a cell cycle-dependent manner and that the hyperphosphorylated forms of BARD1 predominate during M phase. By mobility shift analysis and mass spectrometry, we have identified seven sites of mitotic phosphorylation within BARD1. All sites exist within either an SP or TP sequence, and two sites resemble the consensus motif recognized by cyclin-dependent kinases. To examine the functional consequences of BARD1 phosphorylation, we used a gene targeting knock-in approach to generate isogenic cell lines that express either wild-type or mutant forms of the BARD1 polypeptide. Analysis of these lines in clonogenic survival assays revealed that cells bearing phosphorylation site mutations are hypersensitive to mitomycin C, a genotoxic agent that induces interstrand DNA cross-links. These results implicate BARD1 phosphorylation in the cellular response to DNA damage.

Cell Cycle↗
PubMed2006

Identification of an agent selectively targeting DPC4 (deleted in pancreatic cancer locus 4)-deficient pancreatic cancer cells.

One of the most common types of genetic alterations in cancer is the loss-of-function mutations in tumor-suppressor genes. Such mutations are usually very specific to cancer cells and present attractive and unique opportunities for therapeutic interventions. However, for various reasons, antitumor agents that target loss-of-function mutations have not been readily identified. In this report, using the deleted in pancreatic cancer locus 4 (DPC4) gene in pancreatic cancer as an example, we show the feasibility of a novel screening strategy, which we have named Pharmacological Synthetic Lethal Screening, for the identification of agents that selectively target cancer cells with loss-of-function mutations. We created DPC4 isogenic cell lines through the restoration of wild-type DPC4 in a pancreatic cancer cell line, BxPC-3, whose DPC4 gene was homozygously deleted. The isogenic cell lines were then used in the Pharmacological Synthetic Lethal Screening to evaluate compound libraries for antiproliferative activity and selectivity against DPC4 deficiency. After screening 19,590 compounds, we identified one lead compound, UA62001, which showed 4.6-fold selectivity against DPC4 deficiency in the DPC4 isogenic cell lines. UA62001 selectivity was also seen in another set of DPC4 isogenic cell lines generated by small interfering RNA knockdown. In addition, UA62001 was evaluated in commonly used pancreatic cancer cell lines. A fairly good correlation between DPC4 deficiency and UA62001 sensitivity was observed. Cell cycle analysis indicates that UA62001 arrests cells in S and G(2)-M phases. The results of microarray gene expression profiling and quantitative real-time reverse transcription-PCR suggest that cyclin B/CDC2 and minichromosome maintenance complexes might be the downstream cellular targets of UA62001.

Adenocarcinoma↗
PubMed2003

The intrinsic mitochondrial membrane potential (Deltapsim) is associated with steady-state mitochondrial activity and the extent to which colonic epithelial cells undergo butyrate-mediated growth arrest and apoptosis.

Transformation of colonic epithelial cells is characterized by decreased mitochondrial activity, increased mitochondrial membrane potential (Deltapsi(m)), and disruptions in the equilibrium between cell proliferation and death by apoptosis. We have previously shown that an intact Deltapsi(m) is essential for growth arrest and apoptosis induced by butyrate, a physiological regulator of maturation in these cells, suggesting a role for the Deltapsi(m) in the initiation and integration of proliferation and apoptotic pathways. To extend this work, we have generated isogenic cell lines, from SW620 human colonic carcinoma cells, which exhibit significant differences in intrinsic Deltapsi(m). These differences in Deltapsi(m) are not linked to alterations in viability, Bcl-2 levels, or the differentiation status of the cells. However, compared with parental cells and those with increased Deltapsi(m), cells with decreased intrinsic Deltapsi(m) exhibit significantly higher levels of steady-state mitochondrial mRNA and butyrate-induced p21(WAF1/Cip1) and G(0)-G(1) arrest. Moreover, despite butyrate-mediated translocation of proapoptotic Bax and Bak to the mitochondria, fewer cells with elevated intrinsic Deltapsi(m) exhibit concomitant cytochrome c release, and cells with elevated Deltapsi(m) undergo significantly lower levels of Deltapsi(m) dissipation and apoptosis than parental cells, or cells with decreased Deltapsi(m). Homeostasis of the colonic mucosa depends on balancing cell proliferation with apoptosis, and mitochondrial abnormalities are associated with disruptions in this balance. Thus, by affecting steady-state mitochondrial activity and the extent to which cells enter growth arrest and apoptotic cascades, these data establish a role for the intrinsic Deltapsi(m) in contributing to the probability of colonic tumorigenesis and progression.

Apoptosis↗
PubMed2026

Generation of isogenic gene-corrected cell lines from a USH2A-RP patient-derived iPS cell line.

Comparative studies using induced pluripotent stem cells (iPSCs) from patients with those from healthy individuals as controls are flawed by genetic background contribution to disease phenotype. Here, we used precise gene editing to generate gene-corrected isogenic control lines for a single pathogenic variant in the USH2A gene (c.2276G > T) associated with retinitis pigmentosa (RP). Both homozygously and heterozygously corrected cell lines were successfully generated. These cell lines will serve to unravel RP phenotype differences specific to the USH2A mutation upon their conversion into disease relevant cell types.

Journal Article↗
PubMed2025

Genome editing with programmable base editors in human cells.

Genome editing has garnered significant attention over the last decade, resulting in a massive expansion of the genome engineering toolbox. Base editors encompass a class of tools that enable installing single-nucleotide changes in genomic DNA without the use of double-strand breaks. With the ever-increasing development of new and/or improved base editor systems, it is easy to be overwhelmed by the abundance of options. Here, we provide clear guidance to facilitate the selection of a base editor and to design guide RNAs (gRNAs) to suit various needs. Additionally, we describe in detail how to generate gRNA plasmids, transfect various mammalian cell types, and evaluate editing efficiencies. Finally, we give alternative methods and troubleshooting tips for some common pitfalls encountered during base editing.

Humans↗
PubMed1999

p53 function influences the effect of fractionated radiotherapy on glioblastoma tumors.

PURPOSE: Glioblastoma multiforme brain tumors (GM) are treated with a spectrum of fractionation regimens based on the clinical and anatomical characteristics of the tumor but rarely based on the molecular characteristics of the individual neoplasm. This study tests the hypothesis that the response of cell lines derived from GM to fractionated radiotherapy depends on the function of wild-type p53 (wt p53), a tumor suppressor gene frequently mutated in GM tumors. METHODS & MATERIALS: Isogenic derivatives of glioblastoma cells differing only in p53 function were prepared using a retroviral vector expressing a dominant negative mutant of p53 (mt p53). Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Apoptosis was assayed by a terminal deoxynucleotide transferase-labeling technique and chromatin morphology. RESULTS: We have previously reported the generation of isogenic GM cell lines differing only in p53 function. U87-175.4, lacking wt p53 function, had a significantly lower alpha/beta value than U87-LUX.8, expressing functional wt p53, leading us to hypothesize that fractionated irradiation would preferentially spare GM cells harboring mt p53 compared with those expressing functional, wt p53. Survival curves following either 2.0 Gy or 3.5 Gy/fraction demonstrated that lack of functional wt p53 was associated with resistance to fractionated irradiation. Radiation-induced apoptosis could not account for the observed differences in clonogenic survival. Rather, our data suggested that a deficit in the G1-checkpoint contributed to increased resistance to fractionated irradiation of cells expressing mutant p53. CONCLUSIONS: The effect of fractionated radiotherapy in GM may depend on the function of the tumor suppressor gene p53. A potential clinical consequence of these findings is that hyperfractionation regimens may provide a therapeutic advantage specifically for tumors expressing wt p53 whereas a radiotherapy course of fewer, larger fractions may be appropriate for the treatment of tumors carrying p53 mutations. Further studies are needed to confirm our proposal that the p53 status of GM tumors can be used to guide our choice of fractionation schemes.

Apoptosis↗
PubMed2005

Stability and homogeneity of transgene expression in isogenic cells.

Genetically engineered cells are an important tool not only for basic research applications, but also for biotechnology and molecular medicine. Among the issues yet to be solved for this technology are the consequences of randomly integrating DNA into a cell's genome. The problems encountered range from unpredictable expression levels to safety concerns. Recombinase-mediated chromosome engineering is a popular tool for generating stably transfected isogenic cell lines. Using this approach, single-copy integration of foreign DNA fragments can be achieved at predetermined chromosomal loci in the genome. We used such a technology based on the Flp/Flp recombinase target (FRT) recombination system in human 293 cells for comparative promoter studies. The expected integration patterns were obtained with high frequency. In contrast, the phenotypic characterization of expression of the integrated reporter transgene showed remarkable differences between isogenic cell lines, ranging from homogenous expression to mosaic to even complete expression silencing. As long as cell clones with homogenous expression were kept under selective conditions, their expression characteristics could be maintained over a long period. However, this desirable phenotype was progressively lost upon withdrawal of selective pressure. These results were also reflected by the expression instability of the reporter cassette originally inserted in the recipient cell line. Thus, selection for appropriate integration events that ensure reproducible and long-term gene expression has to go beyond the verification of isogenicity.

Cell Line↗
PubMed2005

Evaluation of the combined effect of p53 codon 72 polymorphism and hotspot mutations in response to anticancer drugs.

Mutations in p53 are common events during carcinogenesis and have been suggested to affect sensitivity to chemotherapy. Recently, the common polymorphism at codon 72, resulting in either an arginine (72R) or proline (72P) residue, was shown to differentially affect the response to anticancer drugs. Here, we have generated isogenic lung cancer cell lines to evaluate the effect of six p53 hotspot mutations (R175H, G245S, R248W, R249S, R273H, and R282W) in conjunction with the codon 72 polymorphism, for their response to a variety of anticancer drugs, either alone or in combination. The data indicate that 72R mutations do not confer general resistance to cisplatin, etoposide, gemcitabine, vinblastine, and taxol. For doxorubicin, cells expressing 249-72R were more resistant than the 249-72P cells. Combined treatment with cisplatin + etoposide resulted in an additive effect in cells expressing most 72R and 72P mutations, except for the 175-72R cells which were refractory to combined treatment. However, combined treatment with cisplatin + gemcitabine resulted in the absence of an additive effect in cells expressing the 273-72R and 282-72R mutants, unlike their 72P counterparts. Nonetheless, all p53 mutants (72R or 72P) equally inhibited p73-mediated transcriptional activity in lung cancer cells, suggesting that the selective resistance conferred by some 72R mutants to certain drugs is probably due to other p73-independent effects of these mutants. Together, the data show that the status of codon 72 polymorphism and p53 mutations can be used as a means for prediction of treatment response, although variables for each cancer type requires detailed evaluation.

Antineoplastic Agents↗
PubMed2026

Establishment of human induced pluripotent stem cell lines and isogenic gene-corrected controls from three patients with prolidase deficiency.

Prolidase deficiency is an autosomal recessive inborn error of metabolism caused by pathogenic variants in the PEPD gene. To date, close to 200 patients have been reported worldwide with a poorly understood pathomechanism. The PEPD gene encodes an enzyme that is involved in the final steps of collagen degradation. Urine amino acid analysis or specific dipeptide analysis can establish the biochemical diagnosis. In this study, we reprogrammed peripheral blood mononuclear cells (PBMCs) from three prolidase deficient patients into induced pluripotent stem cell (iPSC) lines and additionally generated isogenic controls using CRISPR-Cas9 genome editing. The pathogenic PEPD variants identified in our patients were NP_000276.2:p.? (NIHTVBi032-A), NP_000276.2:p.(Ile415Asn)/NP_000276.2:p.(Trp326Ter) (NIHTVBi033-A), and NP_000276.2:p.(Arg265Ter) (NIHTVBi034-A). These iPSC lines are valuable models to help investigate the pathomechanism of prolidase deficiency.

Humans↗
PubMed2006

Growth properties of colonic tumor cells are a function of the intrinsic mitochondrial membrane potential.

Development of malignant transformation in the colonic mucosa includes disruption in the equilibrium between proliferation and apoptosis, decreased expression and deletions of the mitochondrial genome, alterations in mitochondrial enzymatic activity, and elevations in the mitochondrial membrane potential (Deltapsim). Focusing on the role of the Deltapsim in tumor development and progression, we generated novel isogenic colonic carcinoma cell lines that exhibit highly significant, stable differences in their intrinsic Deltapsim. Using these cell lines, we have recently shown that the intrinsic Deltapsim has a significant influence on steady state mitochondrial activity and the extent to which cells enter butyrate-mediated growth arrest and apoptotic cascades. Here, we report that the Deltapsim is also profoundly linked to important tumorigenic properties of the cells. Compared with cells with lower Deltapsim, cells with elevated intrinsic Deltapsim have an enhanced capacity to (a) respond to hypoxia by avoiding apoptosis and initiating angiogenesis, (b) escape anoikis and grow under anchorage-independent conditions, and (c) invade the basement membrane. Combined with our previous work, these data implicate the intrinsic Deltapsim of colonic carcinoma cells in determining the probability of tumor expansion and progression.

Apoptosis↗
PubMed2006

A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species.

The RAS/BRAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is emerging as a key modulator of melanoma initiation and progression. However, a variety of clinical studies indicate that inhibiting the MAPK pathway is insufficient per se to effectively kill melanoma cells. Here, we report on a genetic and pharmacologic approach to identify survival factors responsible for the resistance of melanoma cells to MEK/ERK antagonists. In addition, we describe a new tumor cell-selective means to bypass this resistance in vitro and in vivo. By generating a panel of isogenic cell lines with specific defects in the apoptotic machinery, we found that the ability of melanoma cells to survive in the absence of functional MEK relies on an ERK-independent expression of the antiapoptotic factor Mcl-1 (and to a lesser extent, Bcl-x(L) and Bcl-2). Using computer-based modeling, we developed a novel Bcl-2 homology domain 3 (BH3) mimetic. This compound, named TW-37, is the first rationally designed small molecule with high affinity for Mcl-1, Bcl-x(L), and Bcl-2. Mechanistic analyses of the mode of action of TW-37 showed a synergistic tumor cell killing in the presence of MEK inhibitors. Importantly, TW-37 unveiled an unexpected role of the MAPK pathway in the control of reactive oxygen species (ROS). This function was critical to prevent the activation of proapoptotic functions of p53 in melanoma cells, but surprisingly, it was dispensable for normal melanocytes. Our results suggest that this MAPK-dependent ROS/p53 feedback loop is a point of vulnerability of melanoma cells that can be exploited for rational drug design.

Animals↗
PubMed2026

Changes in the transcriptome and synthetic lethal dependencies following KRAS mutant expression reveal profound tissue specificity.

Oncogenic KRAS mutations exhibit a striking tissue-restricted tropism, occurring with high frequency in pancreatic, colorectal, and lung adenocarcinomas while remaining rare in other lineages. The molecular basis for why these specific tissues are uniquely permissive to KRAS transformation, and how this context shapes therapeutic vulnerabilities, remains poorly defined. Here, we utilized CRISPR-mediated genome engineering to generate endogenous, conditional KRAS-mutant isogenic cell line models across three primary permissive lineages (lung, colon, and pancreas) and the nonpermissive breast lineage. Integrated genome-wide CRISPR fitness screens and comparative transcriptome analyses revealed that KRAS-driven synthetic lethal (SL) dependencies are profoundly shaped by their tissue of origin. Strikingly, we observed minimal overlap in SL hits across lineages, with only three genes shared among the permissive lines, suggesting that the KRAS oncogene operates through divergent, context-specific genetic networks. Mechanistically, we show that KRAS activation induces a universal MYC-driven metabolic signature, but the specific machinery required to sustain this state is lineage-restricted. We identified a dependency on the diphthamide synthesis pathway to maintain translational fidelity amid a KRAS-induced hypertranslational state. These findings demonstrate that even when driven by the same oncogene, tumors exhibit distinct regulatory landscapes and unique genetic vulnerabilities. Our results provide a framework for developing lineage-aware therapeutic strategies, moving beyond universal KRAS inhibition toward targeted interventions tailored to a tumor's specific tissue context.

Proto-Oncogene Proteins p21(ras)↗
PubMed2005

Tumor-host interactions contribute to the elevated expression level of alpha1-antichymotrypsin in metastatic breast tumor xenografts.

We investigated alpha1-antichymotrypsin (ACT) gene expression in xenograft tumors generated by two isogenic human breast cancer cell lines derived from the same parent, MDA-MB-435, which display opposite metastatic behaviors. Microarray and real-time PCR experiments showed an overexpression of this serine protease inhibitor in the metastatic tumors (M-4A4T) and its derived metastases (M4-Mets) compared with the weakly metastatic tumors (NM-2C5T), and its release into the blood was confirmed by western-blotting. However, functional assays in vivo using genetically engineered tumor cells demonstrated that ACT up-regulation was not, by itself, responsible for the metastatic phenotype. We also made observations that ACT gene regulation was sensitive to tumor-host interactions: inoculation of these lines into the mouse mammary gland greatly increased ACT production and accentuated the intrinsic difference observed when they are cultured in vitro. Sensitivity of tumor cells to their environment was further analyzed by in vitro experiments, which demonstrated that a purified ECM environment and soluble components from normal host mammary cells were both able to significantly promote ACT expression. In addition, we took advantage of the xenogeneic nature of the model to measure ACT expression by the host cells (mouse) and the tumor cells (human) within the neoplasm using species-specific primers in real-time PCR experiments. It was found that the presence of tumor cells, irrespective of their metastatic capabilities, induced local ACT production by host cells at the primary and secondary tumor sites. Thus, this work indicates that there is a specific association of ACT overexpression with the metastatic phenotype in our breast cancer metastasis model. Moreover, because of the xenogeneic nature of our system, we were able to provide evidence of tumor-host reciprocal regulation of ACT production.

Animals↗
PubMed2000

Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions.

The retinoblastoma suppressor pRB belongs to the family of so-called pocket proteins, which also includes p107 and p130. These proteins may functionally overlap in cell cycle control and tumor suppression. We have generated an isogenic set of embryonic stem (ES) cell lines carrying single or compound loss-of-function mutations in the Rb gene family, including a cell line completely devoid of all three pocket proteins. None of the knockout combinations affected the growth characteristics of ES cells; however, concomitant ablation of all three pocket proteins strongly impaired their differentiation capacity. For the generated genotypes, primary mouse embryonic fibroblasts (MEFs) also were obtained. While inactivation of Rb alone did not alleviate the senescence response of MEFs, pRB/p107-deficient MEFs, after having adapted to in vitro culturing, continued to proliferate at modest rate. Additional ablation of p130 rendered MEFs completely insensitive to senescence-inducing signals and strongly increased their proliferation rate. Although triple-knockout MEFs retained anchorage dependence, they lacked proper G(1) control and showed increased cell turnover under growth-inhibiting conditions.

Animals↗
PubMed2005

Single inner cell masses yield embryonic stem cell lines differing in lifr expression and their developmental potential.

The unique differentiation potential of inner cell mass derived embryonic stem cells together with their outstanding self-renewal capacity makes them a desirable source for somatic cell therapy of human diseases. Somatic cells are gained by in vitro differentiation of embryonic stem cells, however, the differentiation potential of embryonic stem cells varied even between isogenic cell lines. Variable differentiation potentials may either be a consequence of an inherent inhomogeneity of gene expression in the inner cell mass or may have technical reasons. To understand variations in the differentiation potential, we generated pairs of isogenic, monozygotic twin, and single inner cell mass derived clonal embryonic stem cell lines, and demonstrate that they differentially express the leukaemia inhibitory factor receptor gene. Variations of leukaemia inhibitory factor receptor protein levels are already evident in the inner cell mass and predispose the cardiomyogenic potential of embryonic stem cell lines in a Janus activated kinase dependent manner. Thus, a single inner cell mass may give rise to embryonic stem cell lines with different developmental potentials.

Animals↗
PubMed1996

Protective effect of O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine in DNA and thus may protect cells against genotoxic effects of agents inducing this lesion. To analyze quantitatively the level of protection mediated by MGMT against antineoplastic drugs, we determined the cytotoxic and recombinogenic (sister-chromatid exchange inducing) effects of various chemotherapeutic agents in a pair of isogenic Chinese hamster cell lines deficient and proficient for MGMT, generated upon transfection with human MGMT cDNA. Furthermore, we compared the responses of the human cell lines HeLa MR (MGMT deficient) and HeLa S3 (MGMT proficient) to the various agents. It is shown that: (1) MGMT proficient cells are resistant in cell killing to the methylating drug streptozotocin and all the chloroethylating nitrosoureas tested. There was a marked agent specificity in protection. The level of resistance provoked by MGMT increased in the order BCNU < CCNU < ACNU < HeCNU < streptozotocin. (2) MGMT did not protect cells against killing induced by chlorambucil, cisplatin, melphalan, activated cyclophosphamide (malosfamide) and activated ifosfamide (4-hydroperoxy-ifosfamide). (3) MGMT caused protection against the recombinogenic effect of all nitrosoureas tested. The lowest level of protection was again observed for BCNU, followed by CCNU, ACNU < HeCNU < streptozotocin. (4) MGMT proficient cells did not exhibit resistance in SCE induction towards cyclophosphamide (activated by microsomes), 4-hydroperoxy-ifosfamide, mafosfamide, chlorambucil and melphalan. Some protection was afforded, however, against cisplatin (and transplatin). This effect was abolished by pretreatment of cells with O6-benzylguanine, which depletes MGMT, indicating that some lesion(s) induced by cisplatin giving rise to SCEs can be repaired by MGMT. Taken together, these results indicate that streptozotocin, HeCNU and ACNU are more selective than CCNU and BCNU in killing MGMT deficient cells, and that in the cases of cyclophosphamide, ifosfamide, chlorambucil, cisplatin and melphalan MGMT is not involved in mediating cytotoxic drug resistance.

Animals↗