Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture.

Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity. In this study, we have investigated the feasibility of improving the volumetric productivity by infecting fed-batch cultures at high cell densities. Four commercial and one in-house developed serum-free media were first tested for supporting growth of HEK 293 cells and production of adenovirus type 5 (Ad5) in batch culture. Two best media were then selected for development of fed-batch culture to improve cell growth and virus productivity. A maximum viable cell density up to 16 × 106 cells/mL was achieved in shake flask fed-batch cultures using the selected media and commercial or in-house developed feeds. The volumetric virus productivity was improved by up to six folds, reaching 3.0 × 1010 total viral particles/mL in the fed-batch culture cultivated with the media and feeds developed in house and infected at a cell density of 5 × 106 cells/mL. Additional rounds of optimization of media and feed were required to maintain the improved titer when the fed-batch culture was scaled up in a bench scale (3 L) bioreactor. Overall, the results suggested that fed-batch culture is a simple and feasible process to significantly improve the volumetric productivity of Ad5 through optimization and balance of nutrients in culture media and feeds.

Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus-1.

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 108 TCID50 mL-1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 106 cells mL-1 , and was further improved to 1.1 × 109 TCID50 mL-1 in perfusion culture infected at 4.6 × 106 cells mL-1 . These titers are similar to or better than the previously reported best titer of 8.6 × 107 TCID50 mL-1 and 8.1 × 108 TCID50 mL-1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes.

Repressing expression of difficult-to-express recombinant proteins during the selection process increases productivity of CHO stable pools.

More than half of licensed therapeutic recombinant proteins (r-proteins) are manufactured using constitutively-expressing, stably-transfected Chinese hamster ovary (CHO) clones. While constitutive CHO expression systems have proven their efficacy for the manufacturing of monoclonal antibodies, many next-generation therapeutics such as cytokines and bispecific antibodies as well as biological targets such as ectodomains of transmembrane receptors remain intrinsically challenging to produce. Herein, we exploited a cumate-inducible CHO platform allowing reduced expression of various classes of r-proteins during selection of stable pools. Following stable pool generation, fed-batch productions showed that pools generated without cumate (OFF-pools) were significantly more productive than pools selected in the presence of cumate (ON-pools) for 8 out of the 10 r-proteins tested, including cytokines, G-protein coupled receptors (GPCRs), the HVEM membrane receptor ectodomain, the multifunctional protein High Mobility Group protein B1 (HMGB1), as well as monoclonal and bispecific T-cell engager antibodies. We showed that OFF-pools contain a significantly larger proportion of cells producing high levels of r-proteins and that these cells tend to proliferate faster when expression is turned off, suggesting that r-protein overexpression imposes a metabolic burden on the cells. Cell viability was lower and pool recovery was delayed during selection of ON-pools (mimicking constitutive expression), suggesting that high producers were likely lost or overgrown by faster-growing, low-producing cells. We also observed a correlation between the expression levels of the GPCRs with Binding immunoglobulin Protein, an endoplasmic reticulum (ER) stress marker. Taken together, these data suggest that using an inducible system to minimize r-protein expression during stable CHO pool selection reduces cellular stresses, including ER stress and metabolic burden, leading to pools with greater frequency of high-expressing cells, resulting in improved volumetric productivity.

Culture media selection and feeding strategy for high titer production of a lentiviral vector by stable producer clones cultivated at high cell density.

The growing interest in the use of lentiviral vectors (LVs) for various applications has created a strong demand for large quantities of vectors. To meet the increased demand, we developed a high cell density culture process for production of LV using stable producer clones generated from HEK293 cells, and improved volumetric LV productivity by up to fivefold, reaching a high titer of 8.2 × 107 TU/mL. However, culture media selection and feeding strategy development were not straightforward. The stable producer clone either did not grow or grow to lower cell density in majority of six commercial HEK293 media selected from four manufacturers, although its parental cell line, HEK293 cell, grows robustly in these media. In addition, the LV productivity was only improved up to 53% by increasing cell density from 1 × 106 and 3.8 × 106 cells/mL at induction in batch cultures using two identified top performance media, even these two media supported the clone growth to 5.7 × 106 and 8.1 × 106 cells/mL, respectively. A combination of media and feed from different companies was required to provide diverse nutrients and generate synergetic effect, which supported the clone growing to a higher cell density of 11 × 106 cells/mL and also increasing LV productivity by up to fivefold. This study illustrates that culture media selection and feeding strategy development for a new clone or cell line can be a complex process, due to variable nutritional requirements of a new clone. A combination of diversified culture media and feed provides a broader nutrients and could be used as one fast approach to dramatically improve process performance.

The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions.

Chinese hamster ovary (CHO) cell-based platforms are the most widely used for the biomanufacturing of complex therapeutic proteins, such as monoclonal antibodies (mAbs). The development of high-producing clones that are stable and amenable to large-scale cultures is essential to advance a molecule toward clinical evaluation. Nevertheless, the generation of such clones generally relies on random integration of an expression plasmid encoding the therapeutic protein gene into the host genome. The ensuing clone selection relying on empirical screens and cell line characterization is extensive and time-consuming. An emerging paradigm in CHO cell line development is the use of site-specific recombinases to enable the integration of therapeutic transgenes into pre-marked chromosomal locations with defined expression characteristics. Recombinase-mediated cassette exchange (RMCE) provides a sophisticated alternative to conventional CHO cell line development, leading to the generation of more consistent and reliable clones and may ultimately shorten the "time-to-clinic" of recombinant therapeutics. Herein, we review the recent advances in the use of site-specific recombination systems and their associated cassette exchange technologies for the rapid generation of stable CHO clones with predictable growth, stability, quality and productivity characteristics. Particular emphasis is placed on cassette exchange technologies currently used in the industry. We also discuss the technical hurdles associated with uses of site-specific recombinase systems in CHO cells, illustrate how these problems can be mitigated and provide a perspective on future work concerning these systems.

Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells.

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/l for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change.

Putative chemopreventive molecules can increase Nrf2-regulated cell defense in some human cancer cell lines, resulting in resistance to common cytotoxic therapies.

Nrf2 is a key transcription factor, which induces a cytoprotective gene array. Nrf2 is regulated at the posttranslational level through proteasomal degradation through an interaction with the adapter protein Keap1. High levels of Nrf2, resulting from a loss of function mutation in Keap1, were reported in chemoresistant non-small cell lung cancer. We observed very low levels of Nrf2 and of Nrf2-regulated detoxification proteins as a frequent phenotype in the more chemosensitive breast cancer, and when engineering increased Nrf2 levels, we found resistance to both doxorubicin and paclitaxel. We here show that basal Nrf2 levels in different cell lines correlate with their respective sensitivity to a common cytotoxic chemotherapy. Nrf2 and its regulated genes and proteins are the targets of a major strategy in cancer prevention. Molecules that interfere with the Nrf2-Keap1-Cul3 protein-protein interactions result in higher levels of Nrf2. Both naturally occurring and synthetic molecules with this effect have been suggested as clinical chemopreventive agents, including molecules derived from cruciferous vegetables such as the isothiocyanate sulforaphane and even green tea polyphenols. Here, we determine the impact of these putative chemopreventive agents on the sensitivity of established cancer cell lines to chemotherapy. We confirmed that these molecules do increase Nrf2 and detoxification enzyme levels in breast cancer cell lines with very low basal Nrf2 levels, and this is associated with significant chemoresistance to cytotoxic drugs. Both effects are less in another breast cancer cell line with intermediate Nrf2, and in lung cancer cells with high Nrf2, these same molecules have no effect on Nrf2 but do actually enhance chemoresistance. While the details of dose and schedule of these agents require further study in in vivo models, these data sound a cautionary note for the use of these agents in patients with established cancers who are undergoing chemotherapy treatment.

Cul3 overexpression depletes Nrf2 in breast cancer and is associated with sensitivity to carcinogens, to oxidative stress, and to chemotherapy.

Nrf2 is the key transcription factor for cytoprotective gene programs. Nrf2 is normally maintained at very low concentrations by proteasomal degradation, through its interaction with the adapter protein Keap1 and the Cul3 E3 ligase. Increased Nrf2 concentration resulting from loss of function Keap1 mutations has been described in chemoresistant non-small cell lung cancer. Previous studies in breast cancer showed low levels of some Nrf2-regulated detoxification genes, but the mechanism has not been systematically examined. We found that half of the breast cancer cell lines examined have decreased concentration of Nrf2 compared with normal mammary epithelial cell lines, associated with variable but detectable levels in Keap1 levels, and consistently increased Cul3 mRNA and protein. Immunochemistry showed that 7 of 10 breast cancer specimens examined also have low Nrf2 levels and increased Cul3. Keap1 protein levels are variable. We found no C23Y mutation in Keap1 of any of the cell lines. Using siRNA, we silenced Cul3 in MCF-7 breast cancer cells, and microarray analysis reveals the induction of GCL, NQO1, AKR1C1, UGDH, and TXN by at least 2-fold. The Nrf2-regulated ABCC1 drug transporter was also found to be increased. These Cul3-silenced MCF7 cells are highly resistant to oxidative stress induced by H(2)O(2,) to the carcinogen benzo(a)pyrene, and to both Doxorubicin and Paclitaxel. This high Cul3/low Nrf2 signature may be key to cellular sensitivity to both chemical carcinogeneic stimuli as well as to cytotoxicity of commonly used chemotherapeutic drugs in established breast cancers.

The QKI-6 and QKI-7 RNA binding proteins block proliferation and promote Schwann cell myelination.

BACKGROUND: The quaking viable (qk(v)) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qk(v) mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination. METHODOLOGY/PRINCIPAL FINDINGS: To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27(KIP1) and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27(KIP1) and Krox-20 mRNAs, as assessed by quantitative RT-PCR. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.

Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells.

BACKGROUND: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. RESULTS: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNalpha2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNalpha2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNalpha2b with a recovery greater than 70%. Purified IFNalpha2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNalpha2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNalpha2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNalpha2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNalpha2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNalpha2b produced naturally by lymphocytes. Further, the HEK293-produced IFNalpha2b is biologically active as shown with reporter gene and antiviral assays. CONCLUSION: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNalpha2b.

The bioactivity and receptor affinity of recombinant tagged EGF designed for tissue engineering applications is defined by the nature and position of the tags.

For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure. Our results demonstrate that the type and the location of the tag have a strong impact on growth factor bioactivity (EC50 ranging from 5.5 to 63 nM). Additional surface plasmon resonance-based biosensor experiments were conducted to test the ability of captured chimeric EGF to bind to their receptor ectodomain in vitro. These experiments indicated that the oriented coiled-coil-mediated immobilization of EGF was significantly more efficient than a random approach as coil-tagged EGF displayed enhanced affinities for artificially dimerized EGFR ectodomain when compared to Fc-tagged EGF (apparent KD of 5 pM vs. 16 nM). Altogether, our results highly suggest that coil-tagged chimeras represent an attractive avenue for the oriented immobilization of growth factors for tissue engineering applications and that HEK293 cells offer a robust platform for their expression in a bioactive form.

A sensitive flow cytometry-based nucleotide excision repair assay unexpectedly reveals that mitogen-activated protein kinase signaling does not regulate the removal of UV-induced DNA damage in human cells.

In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.

Novel use of the fluorescent dye 5-(and-6)-chloromethyl SNARF-1 acetate for the measurement of intracellular glutathione in leukemic cells and primary lymphocytes.

Glutathione (GSH) plays an important role in protecting cells against injury, particularly during oxidative stress. Alterations in GSH metabolism are becoming the focus of attention in many diseases such as cancer, neurodegeneration, and AIDS. As such, a rapid assessment of GSH levels in a clinical setting is of increasing importance. We tested the efficacy of the thiol-labeling fluorescent dye CM-SNARF in its ability to measure variations in GSH concentration using a visible-light flow cytometer. GSH levels in I83, Jurkat, and primary lymphocytes were depleted with buthionine sulfoximine (BSO) or diamide, or increased with N-acetylcysteine (NAC). Following each treatment, cells were divided and either labeled with CM-SNARF followed by flow cytometry analysis, or assayed for GSH using a biochemical method. BSO treatment caused a maximal 87-90% decrease in GSH and 68-76% decrease in fluorescence units. Diamide depleted GSH 91-95%, corresponding to a fluorescence decrease of 85-88%. NAC treatment increased GSH levels 27% and fluorescence 12-19%. The overall correlation (R2) between mean GSH concentration and mean fluorescence was 0.80-0.88. CM-SNARF can be used to semi-quantitatively and rapidly determine intracellular variations in GSH concentration in the range of 10-150 nmoles GSH/mg protein.

Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.

XRCC3 depletion induces spontaneous DNA breaks and p53-dependent cell death.

In vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G(2)/M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells. This inhibition of replication coincides with the accumulation of DNA breaks, as shown by the comet assay. Cell cycle specific analysis of gammaH2AX expression shows that S and G2/M phase cells express the highest fraction of gammaH2AX positive cells. This is consistent with replication-dependent accumulation of DNA breaks and deficient homologous recombination. While the induction of gammaH2AX is followed by cell death in parental cells, a p53 knockdown derivative becomes more resistant to XRCC3 depletion-induced death without changes in the levels of gammaH2AX. These results show that XRCC3 is required for the proliferation of MCF7 cells, and that decrease in its expression leads to the accumulation of DNA breaks and the induction of p53-dependent cell death.

FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.

The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

Xrcc3 induces cisplatin resistance by stimulation of Rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis.

Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.

Repression of DNA repair mechanisms in IRF-4-expressing and HTLV-I-infected T lymphocytes.

Human T cell leukemia virus (HTLV) is the causative agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes in which interferon regulatory factor-4 (IRF-4) becomes constitutively expressed, concomitant with major alterations in host gene expression. When constitutively expressed in uninfected T lymphocytes, IRF-4 caused reduced expression of critical DNA repair genes, including Rad51, XRCC1, Ung1, RPA, and proliferative cell nuclear antigen (PCNA), a transcriptional phenotype with striking similarities to the profile observed in HTLV-infected T lymphocytes. Concomitant with the inhibition of gene expression and defects in the DNA repair pathways, increased sensitivity of T lymphocytes to various genotoxic stresses that challenged all major DNA repair pathways were detected. Together, these results support a role for IRF- 4 in the repression of DNA repair activity and an increase in the risk of mutations. IRF-4 may thus represent a previously unidentified endogenous transcriptional repressor of DNA repair mechanisms.

Cdc25A-inhibitory properties and antineoplastic activity of bisperoxovanadium analogues.

Bisperoxovanadium (bpV) compounds are irreversible protein tyrosine phosphatase (PTP) inhibitors with a spectrum of activity distinct from that of vanadium salts. We studied the efficacy of a panel of bpVs as antineoplastic agents in vitro and in vivo with a view to investigating phosphatases as potential antineoplastic targets. The Cdc25A dual-specificity phosphatase is an oncoprotein required for progression through G(1)-S. It cooperates with oncogenic Ras to transform cells and is overexpressed in several cancers. Cdc25A is therefore an attractive candidate phosphatase target for the antineoplastic activity of bpV compounds. Cytotoxicity was examined in 28 cancer cell lines and in vivo efficacy was examined in a DA3 murine mammary carcinoma model. In vitro phosphatase assays were used to directly measure phosphatase inhibition, comparing Cdc25A to hVH2/DSP4, leukocyte antigen related/receptor type PTPF catalytic domain (LAR), Yersinia pestis phosphatase (YOPH), and T-cell PTPase/non-receptor type PTP2 (TCPTP). CDK2 activity and Rb phosphorylation were examined by immunocomplex kinase assays and Western blot. Cdc25A is at least 20-fold more sensitive to bpV inhibition than hVH2/DSP4, and 3- to 10- fold more sensitive than TCPTP and LAR. bpV inhibition of Cdc25A in cells leads to CDK2 inactivation and hypophosphorylation Rb, resulting in G1-S arrest and induction of p53-independent apoptosis. The most cytotoxic analogue, bpV[4,7-dimethyl-1,10-phenanthroline-bisperoxo-oxo-vanadium (Me2Phen)], shows submicromolar IC50s against a panel of cell lines and inhibited tumor growth by 80% in mice. These results demonstrate that bpVs may have significant antineoplastic activity. In addition, they are in vitro and in vivo inhibitors of phosphatases including Cdc25A, suggesting that phosphatases may be appropriate targets for novel antineoplastic agents and that further development of these agents, targeting them to specific phosphatases such as CDC25A, may lead to novel agents with enhanced antineoplastic activity.

The initiation of UV-induced G(1) arrest in human cells is independent of the p53/p21/pRb pathway but can be attenuated through expression of the HPV E7 oncoprotein.

It is well established that the initiation of G(1) arrest in cultured cells exposed to ionizing radiation (IR) is fully dependent upon the p53/p21waf1/pRb signaling cascade. However, the extent to which this pathway regulates G(1) arrest following exposure to UV is less clear. Here we demonstrate that primary human fibroblasts from either skin or lung, in which p53 has been functionally inactivated through expression of the human papillomavirus E6 oncoprotein, each undergoes a prolonged G(1) arrest upon UV irradiation. This same phenomenon is also observed for UV-exposed human tumor cell strains that are genetically deficient for p53, p21waf1 and/or pRb. Furthermore, for the isogenic wild-type counterparts of these primary and tumor cell strains, the onset of UV-induced G(1) arrest precedes any increase in the ratio of hypo- to hyper-phosphorylated pRb and virtually the entire period of growth arrest occurs in the absence of p21waf1 induction. The above data on UV-treated cells are in contrast to the expected situation for IR, for which G(1) arrest is abolished in all deficient cell lines, and, in the wild-type counterparts, correlates precisely with p21waf1 induction and an increase in the ratio of hypo- to hyper-phosphorylated pRb. Remarkably, it was observed that both IR- and UV-induced G(1) arrest are significantly attenuated in primary fibroblasts expressing the human papillomavirus E7 oncoprotein, which functionally inactivates pRb in addition to many other cellular proteins. Our findings conclusively demonstrate that the p53/p21/pRb cascade is not essential for the initiation of G(1) arrest in UV-exposed human cells and, furthermore, indicate the involvement in this process of any among a number of human papillomavirus E7-interacting cellular proteins.