Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections.

Immunogenicity of COVID-eVax Delivered by Electroporation Is Moderately Impacted by Temperature and Molecular Isoforms.

DNA integrity is a key issue in gene therapy and genetic vaccine approaches based on plasmid DNA. In contrast to messenger RNA that requires a controlled cold chain for efficacy, DNA molecules are considered to be more stable. In this study, we challenged this concept by characterizing the immunological response induced by a plasmid DNA vaccine delivered using electroporation. As a model, we used COVID-eVax, a plasmid DNA-based vaccine that targets the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Increased nicked DNA was produced by using either an accelerated stability protocol or a lyophilization protocol. Surprisingly, the immune response induced in vivo was only minimally affected by the percentage of open circular DNA. This result suggests that plasmid DNA vaccines, such as COVID-eVax that have recently completed a phase I clinical trial, retain their efficacy upon storage at higher temperatures, and this feature may facilitate their use in low-/middle-income countries.

A linear DNA encoding the SARS-CoV-2 receptor binding domain elicits potent immune response and neutralizing antibodies in domestic cats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of the COVID-19 pandemic, has been shown to infect a wide range of animal species, especially mammals, and besides human-to-human transmission, human-to-animal transmission has also been observed in some wild animals and pets, especially in cats. It has been demonstrated that cats are permissive to COVID-19 and are susceptible to airborne infections. Given the high transmissibility potential of SARS-CoV-2 to different host species and the close contact between humans and animals, it is crucial to find mechanisms to prevent the transmission chain and reduce the risk of spillover to susceptible species. Here, we show results from a clinical trial conducted in domestic cats to assess safety and immunogenicity of a linear DNA (linDNA) vaccine encoding the receptor-binding domain (RBD) from SARS-CoV-2 (Lin-COVID-eVax). Lin-COVID-eVax proved to be safe, with no significant adverse events, and was able to elicit both RBD-specific antibodies and T cells. Also, the linDNA vaccine induced neutralizing antibody titers against ancestral SARS-CoV-2 virus and its variants. These findings demonstrate the safety and immunogenicity of a genetic vaccine against COVID-19 administered to cats and strongly support the development of vaccines for preventing viral spread in susceptible species, especially those in close contact with humans.

A first-in-human trial on the safety and immunogenicity of COVID-eVax, a cellular response-skewed DNA vaccine against COVID-19.

The COVID-19 pandemic and the need for additional safe, effective, and affordable vaccines gave new impetus into development of vaccine genetic platforms. Here we report the findings from the phase 1, first-in-human, dose-escalation study of COVID-eVax, a DNA vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Sixty-eight healthy adults received two doses of 0.5, 1, or 2 mg 28 days apart, or a single 2-mg dose, via intramuscular injection followed by electroporation, and they were monitored for 6 months. All participants completed the primary safety and immunogenicity assessments after 8 weeks. COVID-eVax was well tolerated, with mainly mild to moderate solicited adverse events (tenderness, pain, bruising, headache, and malaise/fatigue), less frequent after the second dose, and it induced an immune response (binding antibodies and/or T cells) at all prime-boost doses tested in up to 90% of the volunteers at the highest dose. However, the vaccine did not induce neutralizing antibodies, while particularly relevant was the T cell-mediated immunity, with a robust Th1 response. This T cell-skewed immunological response adds significant information to the DNA vaccine platform and should be assessed in further studies for its protective capacity and potential usefulness also in other therapeutic areas, such as oncology.

Persistent B cell memory after SARS-CoV-2 vaccination is functional during breakthrough infections.

Breakthrough SARS-CoV-2 infections in fully vaccinated individuals are considered a consequence of waning immunity. Serum antibodies represent the most measurable outcome of vaccine-induced B cell memory. When antibodies decline, memory B cells are expected to persist and perform their function, preventing clinical disease. We investigated whether BNT162b2 mRNA vaccine induces durable and functional B cell memory in vivo against SARS-CoV-2 3, 6, and 9 months after the second dose in a cohort of health care workers (HCWs). While we observed physiological decline of SARS-CoV-2-specific antibodies, memory B cells persist and increase until 9 months after immunization. HCWs with breakthrough infections had no signs of waning immunity. In 3-4 days, memory B cells responded to SARS-CoV-2 infection by producing high levels of specific antibodies in the serum and anti-Spike IgA in the saliva. Antibodies to the viral nucleoprotein were produced with the slow kinetics typical of the response to a novel antigen.

COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

The Nuts and Bolts of SARS-CoV-2 Spike Receptor-Binding Domain Heterologous Expression.

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

BACKGROUND: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle. Thus, ADP-ribose polymers antagonize the activity of topoisomerase I poisons, whereas PARP inhibitors increase their antitumor effects. METHODS: Using site-directed mutagenesis we have analyzed the interaction of human topoisomerase I and poly(ADP-ribose) through enzymatic activity and binding procedures. RESULTS: Mutations of the human topoisomerase I hydrophobic or charged residues, located on the putative polymer binding sites, are not sufficient to abolish or reduce the binding of the poly(ADP-ribose) to the protein. These results suggest either the presence of additional binding sites or that the mutations are not enough perturbative to destroy the poly(ADP-ribose) interaction, although in one mutant they fully abolish the enzyme activity. CONCLUSIONS: It can be concluded that mutations at the hydrophobic or charged residues of the putative polymer binding sites do not interfere with the ability of poly(ADP-ribose) to antagonize the antitumor activity of topoisomerase I poisons.

Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide.

BACKGROUND: Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. METHODS: Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni's post-test or the non-parametric Kruskal-Wallis analysis and Dunn's post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman's rank test. RESULTS: All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman's test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01). CONCLUSIONS: The combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance.

PARP-1 modulates amyloid beta peptide-induced neuronal damage.

Amyloid beta peptide (Aß) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase (PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aß25-35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aß25-35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aß25-35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aß25-35 induces DNA damage which in turn activates PARP-1. Challenge with Aß25-35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aß25-35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aß and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.

Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion.

The interaction of glucocorticoid-induced tumor necrosis factor receptor-family related (GITR) protein with its ligand (GITRL) modulates different functions, including immune/inflammatory response. These effects are consequent to intracellular signals activated by both GITR and GITRL. Previous results have suggested that lack of GITR expression in GITR(-/-) mice decreases the number of leukocytes within inflamed tissues. We performed experiments to analyze whether the GITRL/GITR system modulates leukocyte adhesion and extravasation. For that purpose, we first evaluated the capability of murine splenocytes to adhere to endothelial cells (EC). Our results indicated that adhesion of GITR(-/-) splenocytes to EC was reduced as compared with wild-type cells, suggesting that GITR plays a role in adhesion and that this effect may be due to GITRL-GITR interaction. Moreover, adhesion was increased when EC were pretreated with an agonist GITR-Fc fusion protein, thus indicating that triggering of GITRL plays a role in adhesion by EC regulation. In a human in vitro model, the adhesion to human EC of HL-60 cells differentiated toward the monocytic lineage was increased by EC pretreatment with agonist GITR-Fc. Conversely, antagonistic anti-GITR and anti-GITRL Ab decreased adhesion, thus further indicating that GITRL triggering increases the EC capability to support leukocyte adhesion. EC treatment with GITR-Fc favored extravasation, as demonstrated by a transmigration assay. Notably, GITRL triggering increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and anti-ICAM-1 and anti-VCAM-1 Abs reversed GITR-Fc effects. Our study demonstrates that GITRL triggering in EC increases leukocyte adhesion and transmigration, suggesting new anti-inflammatory therapeutic approaches based on inhibition of GITRL-GITR interaction.

MSH3 expression does not influence the sensitivity of colon cancer HCT116 cell line to oxaliplatin and poly(ADP-ribose) polymerase (PARP) inhibitor as monotherapy or in combination.

PURPOSE: Defective expression of the mismatch repair protein MSH3 is frequently detected in colon cancer, and down-regulation of its expression was found to decrease sensitivity to platinum compounds or poly(ADP-ribose) polymerase inhibitors (PARPi) monotherapy. We have investigated whether MSH3 transfection in MSH3-deficient colon cancer cells confers resistance to oxaliplatin or PARPi and whether their combination restores chemosensitivity. METHODS: MSH3-deficient/MLH1-proficient colon cancer HCT116(MLH1) cells were transfected with the MSH3 cDNA cloned into the pcDNA3.1(-) vector. MSH3/MLH1-deficient HCT116, carrying MLH1 and MSH3 mutations on chromosome 3 and 5, respectively, and HCT116 in which wild-type MLH1 (HCT116+3), MSH3 (HCT116+5) or both genes (HCT116+3+5) were introduced by chromosome transfer were also tested. Sensitivity to oxaliplatin and to PARPi was evaluated by analysis of clonogenic survival, cell proliferation, apoptosis and cell cycle. RESULTS: MSH3 transfection in HCT116 cells did not confer resistance to oxaliplatin or PARPi monotherapy. MSH3-proficient HCT116+5 or HCT116+3+5 cells, which were more resistant to oxaliplatin and PARPi in comparison with their MSH3-deficient counterparts, expressed higher levels of the nucleotide excision repair ERCC1 and XPF proteins, involved in the resistance to platinum compounds, and lower PARP-1 levels. In all cases, PARPi increased sensitivity to oxaliplatin. CONCLUSIONS: Restoring of MSH3 expression by cDNA transfection, rather than by chromosome transfer, did not affect colon cancer sensitivity to oxaliplatin or PARPi monotherapy; PARP-1 levels seemed to be more crucial for the outcome of PARPi monotherapy.

Influence of MLH1 on colon cancer sensitivity to poly(ADP-ribose) polymerase inhibitor combined with irinotecan.

Poly(ADP-ribose) polymerase inhibitors (PARPi) are currently evaluated in clinical trials in combination with topoisomerase I (Top1) inhibitors against a variety of cancers, including colon carcinoma. Since the mismatch repair component MLH1 is defective in 10-15% of colorectal cancers we have investigated whether MLH1 affects response to the Top1 inhibitor irinotecan, alone or in combination with PARPi. To this end, the colon cancer cell lines HCT116, carrying MLH1 mutations on chromosome 3 and HCT116 in which the wild-type MLH1 gene was replaced via chromosomal transfer (HCT116+3) or by transfection of the corresponding MLH1 cDNA (HCT116 1-2) were used. HCT116 cells or HCT116+3 cells stably silenced for PARP-1 expression were also analysed. The results of in vitro and in vivo experiments indicated that MLH1, together with low levels of Top1, contributed to colon cancer resistance to irinotecan. In the MLH1-proficient cells SN-38, the active metabolite of irinotecan, induced lower levels of DNA damage than in MLH1-deficient cells, as shown by the weaker induction of γ-H2AX and p53 phosphorylation. The presence of MLH1 contributed to induce of prompt Chk1 phosphorylation, restoring G2/M cell cycle checkpoint and repair of DNA damage. On the contrary, in the absence of MLH1, HCT116 cells showed minor Chk1 phosphorylation and underwent apoptosis. Remarkably, inhibition of PARP function by PARPi or by PARP-1 gene silencing always increased the antitumor activity of irinotecan, even in the presence of low PARP-1 expression.

NF-κB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug.

BACKGROUND: Most DNA-damaging chemotherapeutic agents activate the transcription factor nuclear factor κB (NF-κB). However, NF-κB activation can either protect from or contribute to the growth suppressive effects of the agent. We previously showed that the DNA-methylating drug temozolomide (TMZ) activates AKT, a positive modulator of NF-κB, in a mismatch repair (MMR) system-dependent manner. Here we investigated whether NF-κB is activated by TMZ and whether AKT is involved in this molecular event. We also evaluated the functional consequence of inhibiting NF-κB on tumor cell response to TMZ. METHODS: AKT phosphorylation, NF-κB transcriptional activity, IκB-α degradation, NF-κB2/p52 generation, and RelA and NF-κB2/p52 nuclear translocation were investigated in TMZ-treated MMR-deficient (HCT116, 293TLα-) and/or MMR-proficient (HCT116/3-6, 293TLα+, M10) cells. AKT involvement in TMZ-induced activation of NF-κB was addressed in HCT116/3-6 and M10 cells transiently transfected with AKT1-targeting siRNA or using the isogenic MMR-proficient cell lines pUSE2 and KD12, expressing wild type or kinase-dead mutant AKT1. The effects of inhibiting NF-κB on sensitivity to TMZ were investigated in HCT116/3-6 and M10 cells using the NF-κB inhibitor NEMO-binding domain (NBD) peptide or an anti-RelA siRNA. RESULTS: TMZ enhanced NF-κB transcriptional activity, activated AKT, induced IκB-α degradation and RelA nuclear translocation in HCT116/3-6 and M10 but not in HCT116 cells. In M10 cells, TMZ promoted NF-κB2/p52 generation and nuclear translocation and enhanced the secretion of IL-8 and MCP-1. TMZ induced RelA nuclear translocation also in 293TLα+ but not in 293TLα- cells. AKT1 silencing inhibited TMZ-induced IκB-α degradation and NF-κB2/p52 generation. Up-regulation of NF-κB transcriptional activity and nuclear translocation of RelA and NF-κB2/p52 in response to TMZ were impaired in KD12 cells. RelA silencing in HCT116/3-6 and M10 cells increased TMZ-induced growth suppression. In M10 cells NBD peptide reduced basal NF-κB activity, abrogated TMZ-induced up-regulation of NF-κB activity and increased sensitivity to TMZ. In HCT116/3-6 cells, the combined treatment with NBD peptide and TMZ produced additive growth inhibitory effects. CONCLUSION: NF-κB is activated in response to TMZ in a MMR- and AKT-dependent manner and confers protection against drug-induced cell growth inhibition. Our findings suggest that a clinical benefit could be obtained by combining TMZ with NF-κB inhibitors.

Common fragile sites in colon cancer cell lines: role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1.

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.

The glutathione transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) increases temozolomide efficacy against malignant melanoma.

First line treatment of metastatic melanoma includes the methylating agent dacarbazine or its analogue temozolomide (TMZ) with improved pharmacokinetics and tolerability. However, the prognosis of the metastatic disease is poor and several trials are evaluating TMZ in polychemotherapy protocols. The novel glutathione transferase P1-1 (GSTP1-1) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has recently shown activity against melanoma through c-Jun N-terminal kinase activation. In this study we have investigated the in vitro and in vivo efficacy of NBDHEX and TMZ combination against melanoma. The results indicated that NBDHEX and TMZ exerted in vitro synergistic anti-proliferative effects in murine B16 and human A375 melanoma cells. In B16 cells TMZ as single agent caused cell accumulation at the G(2)/M phase of cell cycle, whereas NBDHEX induced mainly apoptotic effects. NBDHEX provoked a higher level of p53 phosphorylation with respect to TMZ and the drug combination caused a more than additive increase of p53 activation. The in vivo efficacy of NBDHEX and TMZ has been investigated in an orthotopic B16 model. Treatment with NBDHEX provoked a reduction of tumour growth comparable to that obtained with TMZ, whereas the drug combination significantly increased tumour growth inhibition with respect to the single agents, without worsening TMZ myelotoxicity. Immunohistochemical analysis of tumour grafts revealed a profound reduction of Cyclin D1 and CD31 in all treatment groups; VEGF expression was, instead, markedly decreased only in NBDHEX or NBDHEX and TMZ treated samples. These findings indicate that NBDHEX represents a good candidate for combination therapies including TMZ, offering new perspectives for the treatment of melanoma.

Poly(ADP-ribose) polymerase signaling of topoisomerase 1-dependent DNA damage in carcinoma cells.

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2(+/+) and p53(+/+). HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT+PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT+PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24-72h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors.

Placenta growth factor induces melanoma resistance to temozolomide through a mechanism that involves the activation of the transcription factor NF-κB.

Placenta growth factor (PlGF) and its receptor vascular endothelial growth factor receptor-1 (VEGFR-1) are co-expressed in a large number of human melanoma cell lines. Moreover, a correlation between in vivo PlGF production and melanoma progression has been suggested. To investigate whether PlGF might have a role in protecting melanoma cells from the cytotoxic effects of the anticancer agent temozolomide (TMZ), which is used for the treatment of this malignancy, we stably transfected a doxycycline-inducible PlGF antisense mRNA into a human melanoma cell clone that secretes VEGF-A and PlGF and expresses receptors for both growth factors. Induction of PlGF antisense mRNA in the transfected cells (13443/ASP3 subclone) halved TMZ IC(50), and exogenous addition of PlGF to the culture medium 24 h before TMZ treatment, partially restored IC(50) values to that of control cells. The increased sensitivity of 13443/ASP3 cells upon PlGF antisense mRNA expression was not due to down-regulation of O6-methylguanine-DNA methyltransferase, a DNA repair protein that represents the main mechanism of resistance to TMZ. Since the activity of the transcription factor nuclear factor-κB (NF-κB) has been correlated to melanoma chemoresistance, we investigated whether NF-κB was involved in PlGF-induced melanoma cell resistance to TMZ. Induction of PlGF antisense mRNA in 13443/ASP3 cells halved the levels of active NF-κB and the specific inhibition of this transcription factor increased sensitivity of 13443/ASP3 cells to TMZ. In conclusion, our data strongly suggest that PlGF plays a role in melanoma cell resistance to TMZ through a pathway that involves NF-κB activation.

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant.

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5' OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.