Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents  and with two isomers of retinoic acid (RA): (9- Treatment of neuroblastoma is multidisciplinary and includes surgery, chemotherapy, radiation therapy, and immunotherapy ,2. ChemoChemotherapeutic protocols in neuroblastoma combine, among others, DNA alkylating agents  and topoisomerase inhibitors (etoposide and doxorubicin). They cause cellular toxicity, eventually leading to DNA double strand breakage, resulting in p53 activation, cell cycle arrest, and induction of apoptosis cell death ,5.The benefit of relatively unconventional biological antitumor agents was first demonstrated in a landmark clinical trial by the Children’s Cancer Group in which the administration of the differentiating agent retinoic acid following recovery from chemotherapy high-dose treatment and stem cell transplantation improved survival for patients with high-risk neuroblastoma . Complethttps://p53.iarc.fr, accessed on 8 July 2021)   . Unlike ly 2021) . Most ofThe MDM2 oncoprotein  is a criA central negative regulator of MDM2 is the tumor suppressor protein p14ARF, which can bind to MDM2 and inhibit its ubiquitin ligase activity, preventing the degradation of p53  and thusAmplification of the MYCN oncogene plays a central role in the pathophysiology and clinical behavior of high-risk neuroblastoma and is associated with an increased vascular index and poor prognosis . Howevercis-RA and all-trans-RA (at-RA)) on the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in neuroblastoma cell lines.Identifying alterations in the p53/MDM2/p14ARF pathway would make it possible to obtain an early molecular diagnosis and a prognostic prediction, and would facilitate the administration of the most appropriate treatments for each neuroblastoma patient. For this reason, the global objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. For that purpose, we have studied TP53 mutations, MYCN and MDM2 amplification, p14ARF methylation, expression, and homozygous deletions, and have analyzed the effects of chemotherapeutic agents  and of two isomers of retinoic acid , SK–N–FI, BE(2)C, IMR–32, Kelly, SIMA, SH–SY5Y, MHH–NB–11, SK–N–MC, and MC–IXC) using MSP (methylation-specific PCR) and MCA-Meth .After treatment with sodium bisulfite, the U primer pair, complementary to the unmethylated DNA sequence, produced amplification of the DNAs A. HoweveObtained results on the expression of the P14ARF gene in the 12 cell lines , SK–N–FI, BE(2)C, IMR–32, Kelly, SIMA, SH–SY5Y, MHH–NB–11, SK–N–MC, and MC–IXC) are shown in p14ARF protein expression in the 12 cell lines was analyzed by Western Blot. Results are shown in Results are shown in The PCR products obtained by amplifying exons 5 to 9 of TP53 were then sequenced in the 12 cell lines , SK–N–FI, BE(2)C, IMR–32, Kelly, SIMA, SH–SY5Y, MHH–NB–11, SK–N–MC, and MC–IXC) looking for possible mutations. Sequence analysis showed a point mutation at the 5′ end of exon 5 in neuroblastoma SK-N-FI cells F. It corFISH (fluorescent in situ hybridization) was carried out to quantify the copy number of the MYCN and MDM2 genes in cell lines SK-N-DZ, SK-N-SH, SK-N-Be(2), SK-N-FI, BE(2)C, IMR-32, Kelly, SIMA, SH-SY5Y, MHH-NB-11, SK-N-MC, and MC-IXC. In the case of MYCN, the RP11-744F11 bacterial artificial chromosome (BAC), labeled with Spectrum Red, was used as a probe; the specific probe for the centromere of chromosome 2 (α2) was labeled with Spectrum Green and used as a reference. To determine the amplification status of the MDM2 gene, the RP11-77H17 BAC was used as a specific probe for MDM2, labeled with Spectrum Red; α12 centromeric probe labeled with Spectrum Green was used as a reference. Results are shown in MDM2 amplification for the BE(2)C, SK-N-DZ, SIMA, and MHH-NB-11 cell lines . These cell lines were selected based on the results obtained with them in the previous sections: mutation of TP53 in SK-N-FI, loss of chromosome 2 in SK-N-FI (aneuploidy), polyploid metaphases in SK-N-Be(2), and homogeneously staining region MYCN amplification in two unknown derived chromosomes in both cell lines. Growth inhibition after 72 h of treatment was determined by the MTT colorimetric assay. Results are represented in dose–response curves for each compound  cells. Therefore, these results evidenced the need to carry out additional experiments to analyze whether the effect of the treatments on cell viability was related to the induction of apoptosis or the modification in the distribution of the cell cycle.Next, the cytotoxicity of the compounds doxorubicin, etoposide, cisplatin, melphalan, 9-compound . As showcis-RA, and at-RA to induce apoptosis in the neuroblastoma cell lines SK–N–FI y SK–N–Be(2), the cells were cultured in DMEM + L-Glutamax supplemented with 10% FBS, 5% non-essential amino acids, and 5 µg/mL of plasmocin. SK-N-FI cells revealed a statistically significant increase in apoptosis levels at 120 h of treatment with each of the compounds, although this increase was already noticeable at 72 h with doxorubicin, cisplatin, and 9-cis-RA  cell line was also determined . The cheIn summary, with the exception of etoposide, the rest of the compounds showed a different capacity to induce apoptosis in cultures depending on the cell line. Thus, while melphalan and doxorubicin were more effective in SK-N-Be(2), cisplatin and the two isoforms of retinoic acid acted more potently in the SK-N-FI cell line, which is mutated in the TP53 gene.cis-RA, and at-RA on the distribution of the cell cycle in SK-N-FI and SK-N-Be(2) cells was estimated by flow cytometry after 12, 24, 72, or 120 h of treatment with the compounds, at concentrations that varied between 0.2 and 1.5 µM depending on the compound used. As previously observed in the apoptosis detection studies, cell cycle distribution was more affected in SK-N-Be(2) cells than in SK-N-FI cells. In all cases except that of at-RA, the first cell cycle alteration observed in both cell lines was an increase in the Sub-G1 phase that, in general, became greater with time.The effect of doxorubicin, etoposide, cisplatin, melphalan, 9-The treatment with doxorubicin at a concentration of 0.2 µM decreased the percentage of cells in the G0/G1 phase, having a more pronounced impact on SK-N-Be(2) cells. Furthermore, in both cases there was an increase of cells in S and G2 phases that seemed to cease at longer times .In relation to the study of treatment with etoposide, we again observed a decrease in the SK-N-FI and SK-N-Be(2) cell population in the G0/G1 phase as time progressed, which was seen more clearly in SK-N-Be(2) cells. We found the same effect for the percentage of cells in phase S. Finally, a blockage in phase G2/M occurred in both cell lines and at all treatment times, although it was more evident in SK-N-Be(2) cells .Treatment with cisplatin at a concentration of 1.5 µM caused a decrease in the percentage of cells in G0/G1 phase in both lines. With respect to the S phase, an increase in the percentage of cells was detected in the SK-N-Be(2) cell line but no changes were observed in SK-N-FI cells. Finally, an increase in cells in the G2/M phase was identified after 72 h of treatment in both cell lines .Regarding the chemotherapeutic agent melphalan, we observed a decrease in the percentage of treated cells in both G0/G1 and S phases at long times, and a blockage of the cell cycle in G2/M only in SK-N-Be(2) cells .Finally, treatment with either of the two isoforms of retinoic acid affected the distribution of the cell cycle only in SK-N-FI cells; a decrease in the percentage of cells in S phase was observed without modifying the percentage of cells in G0/G1 or G2/M phases .Immunofluorescence detection of Bcl-2, p53, and p21 proteins was carried out in SK-N-FI and SK-N-Be(2) cells after 24 h of treatments with the corresponding compounds.Doxorubicin (at 0.2 µM concentration) did not significantly modify the expression of p53, p21, or Bcl-2 in either of the two cell lines analyzed .Etoposide (0.5 µM) did not modify the expression of p53 and p21 proteins, in either of the two cell lines . Howeverp < 0.05) the expression levels of the p53 protein in SK-N-FI cells (p < 0.05). However, this treatment did not modify the expression of these proteins in SK-N-Be(2). The treatment did not affect the levels of p21 in either of the two cell lines.Treatment with cisplatin (1.5 µM) reduced by about 33%  revealed a statistically significant decrease  of the protein in SK-N-FI cells, mutated for TP53. This reduction was not observed in SK-N-Be(2) cells. In the case of p21, practically no alterations were observed, although p21 expression decreased in SK-N-Be(2) cells. Finally, in none of the neuroblastoma cell lines was the expression of Bcl-2 modified significantly as a consequence of the treatment with melphalan .cis-RA (1 µM) gave rise to a clear increase in the expression of p53 in both SK-N-FI and SK-N-Be(2) cells, presenting levels more than two times higher than the control cells. However, there were opposite variations regarding p21 and Bcl-2. A decrease in p21 expression was detected in the SK-N-FI cell line, the opposite effect to that found in SK-N-Be(2) cells. Again, wide variations were estimated for the Bcl-2 protein, whose expression increased in SK-N-FI almost twice and decreased in SK-N-Be(2) to less than half (at-RA (1 µM): a decrease in p53 protein expression was observed in both cell lines, which was statistically significant in the case of SK-N-FI. Treatment also decreased, although not significantly, the levels of p21 in SK-N-Be(2) cells and those of Bcl-2 in both cell types  cells is at least one of the chemo and radioresistance mechanisms in this cell line . FurtherGain of function in some types of tumors is believed to be a mechanism by which mutated TP53 contributes to tumor progression and increased resistance to chemotherapy and ionizing radiation ,40,41. TRetinoic-acid-induced differentiation of neuroblastoma cell lines is associated with a reduction in p53 expression ,42. Treacis-RA in the SK-N-FI cell line that has an inactivating p53 mutation, but not in the SK-N-Be(2) cell line that expresses normal p53.If p53 alterations occur frequently in relapse, or if chemoresistance is associated with loss of p53 function, the use of chemotherapeutic agents in neuroblastoma that either do not depend on the presence of a functional p53  or are more effective if TP53 is inactivated would be highly recommended ,37. Our The lack of accord between RT-PCR data and Western Blot data about p14ARF expression might be clarified by the following hypotheses . MaugeriMethylation of RNA would be another possible explanation for the discrepancies of p14ARF expression data obtained by RT-PCR and Western Blot ,51,52. NCircular RNAs might also be considered as regulators of translation ,54,55,56We analyzed the effect of different antitumor drugs and observed that they have cytotoxic activity at concentrations analogous to those observed in plasma of patients under treatment. Our results show higher apoptosis levels after cisplatin treatment in SK-N-FI cells, despite having mutated TP53, than in SK-N-Be(2) cells.The loss or partial retention of functions and the gain of new activities probably explain the high heterogeneity of cells that express mutated TP53, also with respect to apoptosis. TP53 mutations have been associated with a poor clinical prognosis and resistance to apoptosis-inducing drugs, but proapoptotic activity has also been shown in some cells with mutated TP53 .Paffhausen et al.  suggesteIn our work we have observed MYCN amplification in the SK-N-FI cell line. This could be one reason that explains the greater apoptotic effect of etoposide in these cells compared to the rest of the chemotherapy drugs analyzed. Keshelava et al.  have alsFurthermore, caspase-8-deficient neuroblastoma cells are known to be resistant to apoptosis mediated by death receptors and by doxorubicin . HoweverThe presence of TP53 mutations in cancer cells could not only affect the apoptotic response to treatment but could also allow the cell with genomic damage to enter S phase by preventing cell cycle arrest. In fact, in certain cell types, and in response to some agents that damage DNA, the repair role of p53 could be more important than the induction of apoptosis by p53 . This obDifferent neuroblastoma cell types have been reported to have a decreased ability of p53 to block the cycle in G1, although they express normal and functional p53 and do not have affected apoptosis mediated by p53 . It has cis-RA treatment was accompanied by increases in the expression of p53 that correlated with a greater apoptotic capacity of the cells. Similar results have been described by Xue et al. [In this work we have also observed a lower capacity of cells, both SK-N-FI and SK-N-Be(2), to stop in the G1 phase after treatment with chemotherapeutic agents, a result that occurs without modifications in the expression of p21. However, after treatment with retinoic acid, a differentiating agent, we did not observe changes in the distribution of the cell cycle in any of the cell lines analyzed, despite the fact that 9-e et al. , who havThese authors also observed similar results when treating cells with doxorubicin . RetinoiWe have analyzed whether treatments with antitumor agents of neuroblastoma cell lines that have a functional (SK-N-Be(2)) or mutated (SK-N-FI) TP53 gene modify the levels of p53 protein in these cell lines. Our results show that treatments with doxorubicin and etoposide do not significantly modify p53 levels in either of the two cell lines. Cisplatin and melphalan treatments did not modify p53 levels in SK-N-Be(2) cells, but decreased them in SK-N-FI cells. This decrease could be the consequence of the TP53 mutation in this cell line, manifested with a lower capacity of these cells to stop their growth in G1 and a greater capacity to enter apoptosis. Furthermore, the expression levels of p21 in these cells are not modified by the treatment. This result suggests that the mutation does not completely suppress the transactivating capacity of p53, although it attenuates it, as described in other cases . Despitecis-RA was used and to decreases in p53 expression when at-RA was used. This result may perhaps explain why 9-cis-RA treatment leads to apoptosis, while at-RA treatment preferentially causes cell differentiation. In fact, low levels of p53 expression have been detected in differentiating neuroblastomas and it has been reported that retinoic-acid-induced differentiation of neuroblastoma cells reduces p53 levels in them and that pretreatment of neuroblastoma cell lines with retinoic acid confers on these cells resistance to p53-dependent apoptosis [Interestingly, retinoic acid treatments led to increases in p53 expression when 9-poptosis .It has been suggested that the most frequent event that confers high-level chemoresistance in neuroblastomas is a loss of function of p53 . In thisDoxorubicin treatment has been reported to induce p53-mediated expression of p21 in a mouse model . DoxorubHowever, we detected an inability of cells to stop their growth in the G1/S transition in response to treatments. Dysfunction in the G1 checkpoint of the cell cycle as a consequence of direct mutations of TP53 or of other tumor suppressor genes such as p21 and Rb is widely documented ,28,34,61p53 can also trigger a stop of the cycle in G2/M, through inhibition of CDK1 . It is tcis-RA and in SK-N-Be(2) cells after treatments with at-RA. However, it is noteworthy that 9-cis-RA raised p21 levels in SK-N-Be(2) cells without stopping the cell cycle in G1, which suggests that p21 might not be functional in SK-N-Be(2) cells. Although p21 mutations are rare in human tumors, those that have been identified appear to be incapable of inhibiting CDK activity and blocking the cell cycle in G1. Possible alternatives to explain the attenuation of G1 arrest include amplification of cyclin D1, cdk4, cdk6, and pRb mutations. These possible alterations have been described in neuroblastoma, although their frequency is low [We did not detect alterations in the cell cycle after treatment with the different isomers of retinoic acid. A decrease in p21 was observed in SK-N-FI cells after treatments with 9-y is low .Bcl-2 is strongly expressed in most primary tumors and neuroblastoma cell lines, and its expression levels are inversely correlated with the proportion of cells that undergo apoptosis and with the degree of cell differentiation . It has We have observed a decrease in the levels of Bcl-2 expression that correlates with an increase in apoptosis in the SK-N-FI cell line after 24 h of treatment with cisplatin and/or etoposide. This result supports the idea that p53 functions, despite the mutation of the protein, are conserved, at least partially, in this cell line. It also suggests the correct functioning of the intrinsic pathway of apoptosis in these cells. On the contrary, the reduction in the expression levels of Bcl-2 without induction of apoptosis observed in SK-N-Be(2) cells after treatment with retinoic acid suggests a defect in the intrinsic pathway of apoptosis. The regulation of apoptosis is another possibility to explore in targeted therapies: the analyses of the effect of G3139, an antisense of Bcl-2, and of obatoclax, a pan-inhibitor of the Bcl-2 family, are current examples of these analyses [https://www.atcc.org/products/crl-2270#detailed-product-information, accessed on 11 May 2010). These two cell lines were considered to be neuroblastoma cells some years ago. Finally, the glioma cell line SW1783  was used as a positive control for the expression of the p14ARF protein.A panel of 10 neuroblastoma cell lines established from patients at different stages of therapy and the glioma cell line SW1783 as a control were used. Five of them , SK-N-FI, and Be(2)C) were obtained from the American Type Culture Collection , while the remaining five  were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH . SK-N-MC  and MC-IXC  cell lines were also studied. SK-N-MC is a neuroepithelioma cell line that today is considered to be derived from an Askin’s tumor, morphologically similar, related to Ewing’s sarcoma . MC-IXC 2 humidified incubator. In addition, 1% penicillin/streptomycin as antibiotic  and 0.1% amphotericin B as antifungal agent  were added. SW1783 glioma cell line was grown in RPMI L-Glutamax , 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin , and 0,1% amphotericin B .The 10 neuroblastoma cell lines, SK-N-MC, and MC-IXC were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% heat-inactivated fetal bovine serum  and 5% non-essential amino acids  in a 37 °C, 5% CODNA from the cell lines was purified by the Wizard Genomic DNA Purification Kit  according to manufacturer’s instructions. In addition, 1 μg genomic DNA was bisulphite modified by the CpGenome™ DNA Modification Kit , following the manufacturer’s protocol. Briefly, genomic DNA was denatured by NaOH with a final concentration of 420 mM at 37 °C for 10 min. DNA was modified by adding 550 μL of 3M sodium bisulphite (pH 5.0) and incubating it for 16 h at 50 °C. The bisulphite-treated DNA was washed with 90% ethanol, precipitated, resuspended in 1 mM TE pH 8, and stored at −80 °C until use. Peripheral blood genomic DNA from a healthy donor and in vitro methylated DNA  were used as negative and positive controls, respectively, for the methylation status of DNA.2, 0.2 mM of each dNTP, 15 pmol forward and reverse primers, 5% DMSO, and 1 U BioTaq™ DNA polymerase . PCR reactions were carried out in a PTC-200 Peltier Thermal Cycler . PCR reactions were denatured at 95 °C for 11 min, followed by 35 cycles of 30 s at 94 °C, 30 s at the annealing temperature of 62 °C, 30 s at 72 °C, followed by a final extension step at 72 °C for 10 min. PCR products were visualized in a 2% agarose gel stained with ethidium bromide at a final concentration of 0.1 μg/mL.For the analysis of the methylation pattern in the CpG islands of the promoter of the P14ARF gene we used methylation-specific PCR (MSP). Bisulphite-modified DNA was amplified by PCR using two primer sets specific for methylated (M) and unmethylated (U) sequences of the p14ARF gene previously described . The metMelting curve analysis–methylation assay (MCA-Meth) was performed with 6 ng of bisulphite modified DNA in a total volume of 25 μL, containing 12.5 μL of Brilliant SYBR Green Master Mix  and 2.5 pmol of primers, in an IQ5 Multicolor Real-Time PCR Detection System . MCA-Meth primers were designed using the MethPrimer software , lackingRNA was extracted from cell cultures using the RNeasy Mini Kit , according to the manufacturer’s instructions. Possible contaminating DNA was removed with the Amplification Grade DNase I . Total RNA (1 μg) was transcribed to cDNA by using the Superscript II kit .2, 5% DMSO, 10 pmol forward and reverse primers for p14ARF gene (5 pmol for TFR), 75 ng of cDNA, and 1 U Amplitaq Gold™  in a final volume of 25 μL. The reaction conditions of 95 °C for 10 min, 43 cycles of 1 min denaturation at 94 °C, 45 s annealing at 62 °C, 45 s extension at 72 °C, and final extension step for 10 min at 72 °C were used to amplify p14ARF. For TFR, the reactions were cycled with a 10 min initial denaturation step at 95 °C, followed by 30 cycles of 45 s denaturation at 94 °C, 45 s annealing at 60 °C, 2 min extension at 72 °C, and 10 min final extension at 72 °C. PCR products were loaded in an ethidium-bromide-stained 2.5% agarose gel and were subjected to electrophoresis.A fragment of 298 bp of the transferrin receptor (TFR) gene was previously amplified as a control of RNA integrity. Primers used for TFR and p14ARF expression study are described in Briefly, neuroblastoma cell line pellets were resuspended in ice-cold RIPA cell lysis buffer , supplemented with 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, 0,1% sodium deoxycholate, and 0,1% proteinase inhibitor cocktail  at pH 7.2. Proteins were quantified following the BCA Protein Assay-Reducing Agent Compatible kit  by using a spectrophotometer.First, 20–30 μg of proteins was separated by SDS-PAGE in 15% polyacrylamide gels, transferred to polyvinylidene difluoride membranes , and incubated with primary antibody anti-p14ARF  and peroxidase conjugate secondary anti mouse antibody IgG . Immunoblots were then visualized by using enhanced chemiluminescence ECL . Protein values obtained were normalized regarding the amount of GAPDH.2, 0.2 mM dNTPs, 10× buffer, 5 pmol of the corresponding pair of primers for p14ARF and GAPDH, respectively, and 1 U of BioTaq™ DNA polymerase . In each PCR, a negative control for homozygous deletions of p14ARF  was also included to calculate the normal dosage of the target gene. The PCR products were loaded onto ethidium-bromide-stained 15% polyacrylamide gels (19:1) to obtain a higher resolution and therefore be able to differentiate between the bands of each gene.Differential polymerase chain reaction assays were performed to study homozygous deletions of p14ARF (exon 1-β). A fragment of the GAPDH gene was used as an internal control. The sequences of the primers used are shown in 2, 10 pmol forward and reverse primers, 50 ng DNA, and 1 U BioTaq™ DNA Polymerase  in a final volume of 25 μL. Thermal conditions were 30 cycles of 94 °C for 45 s, 65 °C for 45 s, and 72 °C for 45 s, with an initial 10 min denaturation at 95 °C and a final 10 min extension at 72 °C. Control samples were also included in the sequencing of all exons. The PCR products were gel-purified with columns,  and sequenced in both directions at CIMA, University of Navarra.Genomic DNA extracted from the cell lines was used for sequencing analysis. TP53 exons 5 to 9 were amplified by PCR using primers described by Toguchida et al. , whose shttps://genome.ucsc.edu, accessed on 11 May 2010) or deposited in GenBank (accession no. X54156 and U94788) using EMBOSS Pairwise Alignment Algorithms tool.TP53 sequence results were compared to the reference wild-type sequences from the Genome Browser of the University of California in Santa Cruz  and incubated in the oven at 37 °C for 2 or 3 h. The medium was then collected in a centrifuge tube, and the trypsin was neutralized once the cells, already washed with PBS, had detached from the flask. After centrifuging and discarding the supernatant with the vacuum pump, a hypotonic shock was caused by slowly adding 5 mL of a 75 mM KCl solution previously heated to 37 °C . After centrifuging, the supernatant was removed, except for 1 mL, which was homogenized in the vortex and to which 5 mL of 4:1 Carnoy’s fixative (methanol/acetic acid) was added dropwise. The tubes were kept for 10 min on ice before re-centrifuging and removing the supernatant. The fixation process was repeated three times with methanol/acetic acid (3:1), before resuspending the cells and making the extensions on dry, clean slides previously cooled in a refrigerator. The preparations were stored at −20 °C.To obtain metaphase cytogenetic suspensions of cell lines, mitoses of cells in culture were stopped by adding a volume of 180–260 µL of colchicine solution with a concentration of 10 µg⁄mL  and MDM2 on chromosome 12 (12q14.3-q15) were chosen. The centromeric probes were labelled with Spectrum Green fluorochrome and those of MYCN and MDM2 with Spectrum Red. Hybridizations were performed with a pair of centromere/gene probes simultaneously, so that in each of the cell lines a double fluorescent labelling could be produced: the centromeres in green and the MYCN and MDM2 genes in red.https://genome.ucsc.edu, accessed 11 May 2010) allowed the selection of different BACs that comprised 100–200 Kb fragments of DNA, large enough to be used as probes in FISH studies.For the localization of genomic clones that covered the regions of interest, the Genome Browser of the University of California in Santa Cruz .For the study of the MYCN gene, the BACs RP11-744F11, RP11-120J4, and RP11-635A14 were selected, while for the BACs that overlapped the E.coli (BACs) are supplied on unstable LB agar which limits their viability. A culture was made in solid LB medium  supplemented with chloramphenicol 12.5 µg/mL by loop depletion seeding, at 37 °C overnight. To obtain the DNA, one of the isolated colonies was selected and amplified by growing it in 10 mL of liquid LB supplemented with chloramphenicol, overnight, at 37 °C while shaking at 250 rpm. The plasmids corresponding to the α2 and α12 centromeric probes were amplified by growing them in 12 mL of LB medium supplemented with ampicillin at a concentration of 25 µg/µL at 37 °C, under constant shaking (250 rpm), overnight. For the conservation of the BAC or plasmid clones, they were frozen at −80 °C in LB-glycerol (800 µL of liquid culture and 200 µL of sterile glycerol). Successive cultures were made from these reservoirs. After plasmid miniprep and determination of the DNA concentration and quality, the labelling of the probes and their subsequent hybridization with the cytogenetic suspensions of the cell lines were subcontracted to the company Genetadi Biotech SL , who counted the signals of 100–200 nuclei from each cell line on a Leica DM6000 microscope.Bacterial clones in MYCN FISH establish that gene amplification is defined as an increase in MYCN signal greater than four times with respect to the number of chromosomes 2. In turn, gene gain refers to an excess of two to four times in the copy number of MYCN with respect to chromosome 2 [In neuroblastoma, the published guidelines for mosome 2 . Accordimosome 2 ,2): colomosome 2 ,4): coloThe count of the predominant clones in the BE(2)C, IMR-32 and SK-N-Be(2) cell lines showed two signals of α2 and multiple signals corresponding to RP11-744F11 (2G ampR) which indicates amplification of MYCN on these lines (H-tetrazolium bromide) developed by Mosmann in 1983 [Cell viability of the neuroblastoma lines SK-N-FI and SK-N-Be(2) was quantified by the MTT colorimetric assay -2,5-diphenyl-2 in 1983 . Apoptoscis-RA (1 µM), or at-RA (1 µM). After the treatments, the cells were trypsinized, collecting the apoptotic bodies from the supernatant by centrifugation. According to the manufacturer’s instructions, they were resuspended for 1 h at 4 °C in 1% (w/v) paraformaldehyde  in PBS pH 7.4  at a concentration of 1 × 106 cells/mL. After centrifuging for 5 min at 300× g and removing the supernatant, the cells were washed twice with 5 mL of PBS and the pellet was resuspended in the residual PBS by vortexing the tube. Finally, the cells were fixed in ice-cold 70% ethanol adjusting the concentration to 1 × 106 cells/mL, incubated for at least 30 min at 4 °C, and stored at −20 °C.The ability of the different drugs to induce apoptosis in SK-N-FI and SK-N-Be(2) cells was determined after 12, 24, or 72 h or 5 days of treatment with doxorubicin (0.2 µM), etoposide (0.5 µM), cisplatin (1.5 µM), melphalan (0.3 µM), 9-6 cells/mL) for 5 min at 300× g. After washing the cells twice, they were incubated at 37 °C for 1 h with the labelling solution, which contains the TdT enzyme, FITC-dUTP, and reaction buffer. Rinse solution was added and centrifuged again for 5 min at 300× g. This process was repeated by removing the supernatant by aspiration. Finally, the precipitate was resuspended in 0.3–0.5 mL of propidium iodide/RNase solution and incubated for 30 min in darkness at room temperature.For labelling, residual ethanol was removed from the supernatant after centrifuging the fixed cells , equipped with an argon laser (488 nm). The data, on a logarithmic scale, were analyzed with the EXPO32 ADC software . Cytometer calibration was checked in each experiment using Flow-Check™ Fluorospheres (Beckman Coulter).Propidium iodide marks the total DNA of the cells and emits fluorescence at 623 nm, providing additional information on the DNA content and therefore on the phase of the cell cycle in which each cell is. The histogram recording the fluorescence of propidium iodide for DNA content analysis  was visualized with the EXPO32 ADC software on 20,000 cells.Rabbit polyclonal primary antibodies directed against the N-terminal ends of the Bcl-2 (N-19) and p21 (N-20) proteins and a goat polyclonal primary antibody to detect the C-terminal end of p53 (C-19)  were used. Isotype antibodies of the different phosphorylated proteins  were used as negative controls. Subsequently, the experiment was continued by incubation with secondary antibodies bound to FITC, of goat in the case of Bcl-2 and p21, and of donkey in that of p53 . The samples, in the dark and at 4 °C, were analyzed in the cytometer with no more than 4 h having elapsed since the labeling. Expression analysis was determined from 10,000 cells per sample. The mean fluorescence obtained was subtracted from that corresponding to its negative control to eliminate the nonspecific labeling. At least three independent experiments were carried out in duplicate.The distribution of samples in apoptosis, cell cycle, and protein expression studies between treated and control cells was analyzed using the Shapiro–Wilks and Kolmogorov–Smirnov tests for normality. Analyses were carried out with the statistical program SPSS 16.0 for Windows . In the groups in which these tests were significant, a Mann–Whitney U test was performed for non-parametric samples, while in the groups whose distribution was normal, the Student’s t test was performed for parametric samples. The significance limit of the tests was set at 0.05.In summary, we have characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway, finding that no cell line presented MDM2 amplification, p14ARF promoter methylation, p14ARF homozygous deletions, or p14ARF expression, while only one of them had a TP53 point mutation. We have also treated two of those cell lines with chemotherapeutic agents  and with two isomers of retinoic acid. SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cells responded differently to the retinoid isomers. The mutation at TP53 might confer a better response capacity to apoptosis and to stop cell cycle progression, all of which makes us rethink the necessity of deciphering the molecular status of TP53 prior to treating patients of neuroblastoma in order to standardize therapies with the aim of improving survival. As seen here, TP53 does not follow a unique pattern of behavior in cancer cells. Strategies to restore normal function in TP53 mutated tumors, like those of APR-246 (PRIMA-1met) ,77,78,79


Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN. The selective filtration of plasma is performed in the cortex of the kidney in the glomerulus of the nephron. Endothelial cells of blood arteries that enter the glomerulus, a glomerular basement membrane (GBM) between them and podocytes, and podocytes themselves form the glomerular filtration barrier (GFB). Podocytes are highly specialized cells that are dedicated to plasma filtration. They consist of a cell body and major and foot processes. Foot processes branch out from major processes and tightly cover blood arteries in the glomerulus. Between neighboring foot processes, a cell–cell junction is present, forming slit diaphragms that form a size-selective barrier for the excretion of proteins that is critical for proper kidney filtration .Diabetes mellitus (DM) affected 8.5% of adults worldwide in 2014 . It is cKlotho is an anti-aging molecule. Upon its discovery, Klotho was shown to extend its lifespan in mice by 30% and protect them from many disorders, especially disorders of renal tissue ,14. KlotKlotho has frequently been reported to decrease inflammation through suppression of the activation of numerous proinflammatory cytokines, chemoattractants, and receptors. Moreover, Klotho inhibits tissue fibrosis ,19. TherBoth membrane-bound and soluble Klotho can bind to fibroblast growth factor (FGF) receptors 1-4 (FGFR1-4), serving as a coreceptor protein for FGF23. Klotho alters the structure of these receptors and increases their binding affinity for FGF23 twenty-fold. FGFRs are widespread throughout the body ,25. HoweThe present study analyzed levels of Klotho expression in renal tissue and its levels in serum and urine under both standard and hyperglycemic conditions. Additionally, we investigated whether Klotho influences FGFR1-4 expression and glucose metabolism in podocyte cells that are cultured in media with standard glucose (SG) or high glucose (HG) concentrations. Finally, we investigated whether Klotho protein influences albumin permeability of the GFB. In brief, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN.11 ± 3.47 × 1010 nm2 vs. 1.37 × 1012 ± 9.36 × 1010 nm2, respectively; During acute kidney injury, both plasma and urine levels of soluble Klotho protein decrease in affected individuals, which was proposed to be a biomarker of the disease . TherefoThe amount of soluble Klotho protein decreased in urine, and membrane-bound Klotho decreased in renal tissue and glomeruli in diabetic rats . We nextFGFR1 and FGFR2 genes significantly decreased by 20% and 21.5%, respectively , >98% decrease in FGFR2 (1.85 × 1010 ± 1.56 × 109 nm2 vs. 9.87 × 1011 ± 1.09 × 1011 nm2), ~83% decrease in FGFR3 (2.23 × 1011 ± 8.44 × 109 nm2 vs. 1.31 × 1012 ± 3.34 × 1010 nm2), and >78% decrease in FGFR4  caused a ~15% and ~18% increase in the mRNA expression of FGFR1 and FGFR2, respectively, in human podocytes that were grown under SG conditions , low levels of calcitriol are found, which is an activator of Klotho expression . We founKim et al., reported the presence of Klotho in human glomeruli . To our FGFR1 and FGFR2. We also found that the addition of recombinant Klotho to the cell medium increased the mRNA expression of FGFR1 and FGFR2 under SG conditions, with an upward tendency under hyperglycemic conditions. This tendency accelerated at the protein level, in which the addition of Klotho significantly increased the expression of both FGFR1 and FGFR2 in podocytes that were grown in HG, which was also confirmed by immunofluorescent staining.We also found that all four FGFRs were present in human podocytes at both the mRNA and protein levels. Under hyperglycemic conditions, we observed a significant decrease in the protein levels of FGFR1-4 and the mRNA expression of Studies of the level and function of FGFRs in renal tissue in diabetic individuals are scarce. Consistent with the present findings, Taylor et al., reported a decrease in the protein level of FGFR4 in mice with CKD . Cheng eIn the present study, Klotho increased glycolysis and glycolytic capacity in human podocytes under both standard and hyperglycemic conditions. To our knowledge, the only studies that investigated the influence of Klotho protein at the level of glycolysis were performed under aerobic conditions and with organs other than the kidneys. In the brain, full-length Klotho protein participates in supplying nutrients to neurons by astrocytes. Klotho stimulates a rapid increase in aerobic glycolysis and lactate release from astrocytes, which is needed for energy production by neurons . HoweverOur research group previously found that HG conditions increase albumin permeability in rat glomeruli and cultured rat podocytes, accompanied by F-actin redistribution to cortical regions of podocytes ,48,49. HTo strengthen the findings of the current study, future analyses should concern testing of human serum and/or urine samples for Klotho level assessment. These analyses could also show results of iv vivo-based studies of FGF receptors levels, glycolytic parameters and albumin permeability of glomeruli and podocytes after Klotho supplementation in diabetic rats.The experiments were performed with male Wistar rats (167–342 g) that were obtained from the Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland. The rats were maintained on a 12 h/12 h light/dark cycle with free access to a standard pellet diet and tap water. The experiments were conducted in accordance with directive 2010/63/EU, and the protocol was approved by the local Ethical Commission at the University of Science and Technology in Bydgoszcz, Poland (no. 54/2018).The experiments were conducted with male rats that were subjected to diabetes and induced by streptozotocin , and age-matched control Wistar rats. In diabetic rats, the experiments were performed 14 days after diabetes induction, only in animals with fasting blood glucose concentrations >250 mg/dL . Blood s2, 0.9 mM CaCl2, and 5.6 mM glucose. The renal capsule was removed, and a cortex was minced with a razor blade and then pressed through a series of sieves with decreasing pore diameters . The obtained cell suspension contained decapsulated glomeruli without afferent and efferent arterioles. The entire procedure was performed in an ice bath and completed in less than 1 h.Male Wistar rat kidneys were removed and placed in ice-cold phosphate-buffered saline  supplemented with 0.49 mM MgClHuman immortalized podocytes  were generated, as described previously . The expTo assess the amount of membrane Klotho protein in human podocytes, cells were incubated for 30 min with biotin solution  in PBS (pH 8.0). Excess biotin was washed out with PBS with the addition of glycine . Subsequently, the cells were lysed, as described previously . A portiHuman podocytes and rat glomerulus lysates were prepared, as described previously ,52. AmicFormalin-fixed paraffin-embedded kidney specimens that were obtained from Wistar control and Wistar STZ rats were deparaffined with Histochoice Cleaning Agent (Sigma Aldrich) and ethanol and hydrated in water. Epitopes were retrieved by incubating the specimens in sodium citrate buffer  at 97 °C for 20 min. The specimens were blocked with 5% bovine serum albumin (BSA) for 1 h. Bovine serum albumin was then removed from the slides, and primary antibodies (diluted in 5% BSA) were added to the specimens, followed by overnight incubation at 4 °C . The nexhttps://myassays.com/) (accessed on 3 August 2020).A commercially available Rat Klotho ELISA Kit  was used to determine concentrations of soluble Klotho in serum and urine samples from Wistar rats. Twenty-four hour urinary Klotho excretion was calculated based on urine Klotho concentration and the volume of 24-h urine excretion. The assay was performed in accordance with the manufacturer’s instructions. Data was analyzed using MyAssay software . Quantity and purity of the obtained RNA were determined using a NanoDrop device (Thermo Fisher Scientific). A reverse transcription reaction was performed on isolated RNA. The obtained complementary DNA was subjected to a real-time polymerase chain reaction (PCR) that was performed using a LightCycler 480 instrument  with gene-specific intron-spanning primers and 8 bp fluorescent hydrolysis probes Roche; . RelativPodocytes were grown on glass coverslips that were coated with type I collagen. After incubation under experimental conditions, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X 100, and blocked with 3% BSA in PBS (blocking solution). Subsequently, the cells were incubated overnight at 4 °C with primary antibodies  that werA glycolysis stress test was performed using a Seahorse XFp analyzer , which depends on measurements of the extracellular acidification rate (ECAR). The ECAR increases through proton release from cells to the assay medium, which occurs during the glycolysis process. Glycolytic parameters that are obtained in the analysis are mainly levels of glycolysis and glycolytic capacity . First, 2, 0.9 mM CaCl2, and 5.6 mM glucose) that contained 5% BSA. Adherent glomeruli were then incubated in 5% BSA medium that contained Klotho protein (0.5 nM) for 30 min at 37 °C. The initial incubation medium was replaced with a medium that contained 1% BSA to produce an oncotic gradient across the glomerular capillary wall (5% BSA in the lumen vs. 1% BSA in the bath medium). Control glomeruli were treated with equivalent volumes of medium that contained 5% BSA (no oncotic gradient). Glomerular volume changes were recorded by video microscopy  before and after a medium change to an oncopressive medium (1% BSA). Glomerular volume (V) was calculated from the surface area (A) of the glomerulus using the following formula using Olympus CellSens Dimension software: V = (4/3A6. Volume changes (ΔV) were calculated as ΔV = /Vinitial based on the direct relationship between the increase in ΔV and oncotic gradient that was applied across the capillary wall. This principle was used to calculate the reflection coefficient of albumin , defined as the ratio of ΔV for experimental glomeruli to ΔV of control glomeruli in response to identical oncotic gradients, where σalb = ΔVexperimental/ΔVcontrol. The reflection coefficient of albumin  was used to calculate the glomerular capillary permeability to albumin , which describes the movement of albumin consequent to water flow. The volume response of glomerular capillaries to an oncotic gradient that was generated by defined concentrations of albumin was measured, as described previously . BrieflyThe transepithelial albumin permeability of human podocyte cells was assessed by measuring FITC-labeled BSA diffusion through a podocyte monolayer, as previously described , with miX-axis at a 1 μm distance is expressed as a percentage of the mean value of the fluorescence intensity that was calculated for the total X-axis. The cell membrane was positioned at point 0 on the plot.To visualize the F-actin network in human podocytes that were cultured on human fibronectin-coated coverslips, Alexa Fluor 633 Phalloidin  was used. NIS-Elements General Analysis 3 software (Nikon) was used to generate fluorescence intensity profiles from the basal membrane to the nucleus. The normalization of fluorescent profiles of different cells was performed. The fluorescence intensity on the post hoc test, unpaired t-test) or nonparametric tests . Values of p ≤ 0.05 were considered statistically significant. The results are expressed as mean ± SEM.The data were analyzed using Prism 5.03 software . Datasets were tested for a Gaussian distribution using the Shapiro–Wilk test. Depending on the results of this test, the statistical significance of the data was analyzed using parametric tests  followed by Bonferroni’s multiple-comparison In conclusion, we observed significantly higher serum levels of soluble Klotho and a decrease in its urinary excretion, which may be considered early biomarkers of DN. We found that Klotho improved the function of renal tissue through effects on the restoration of FGFRs, improved glycolysis, and lowered albumin permeability under hyperglycemic conditions. Our findings indicate that Klotho should be investigated further with regard to its potential role in reducing the pathological effects of DN.


BRCA1 or BRCA2 deficiency and in their BRCA1/2-proficient counterparts. Patients with tumors positive for MET had worse overall survival  compared to patients with MET-negative tumors. The prognostic role of MET was even more prominent in the subgroup of patients with SOX2-negative tumors (p = 0.0081). No synergistic effects of the combined treatment with PARP and MET inhibitors were found in the cell lines examined. We conclude that MET expression could be used as a marker for OS in HGSC and that stemness should be taken into consideration when evaluating the mechanisms of this effect.Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several cancer types, and MET has been suggested to interact with stem cell networks. In vitro studies have further suggested a possible benefit of a combined treatment using PARP and MET inhibitors. We used a tissue microarray (TMA) with 130 samples of advanced-stage high-grade serous fallopian tube/ovarian cancer (HGSC) to investigate the prognostic value of MET protein expression alone and in combination with the stem cell factor SOX2. The possible synergistic effects of a PARP and MET inhibitor treatment were evaluated in two cell lines with  BRCA1/2 mutation is present (either germline or somatic) [BRCA1/2 mutation status, again requiring at least a partial response to re-treatment with platinum [In 2020, 314,000 women worldwide were diagnosed with ovarian or fallopian tube cancer. The disease accounted for 3.4% of all female cancer cases and 4.7% of all cancer-related deaths in women [platinum ,6. To daBRCA1 and BRCA2, but PARPi also show effect in ovarian cancer patients without BRCA1/2 mutations. Overall survival (OS) rates have been shown to increase in both BRCA1/2-mutant and -wildtype carriers following PARPi treatment, provided that the tumors are still platinum-sensitive [The response to PARPi is facilitated through binding of the drug to PARP proteins, thereby disrupting the repair of single-strand breaks. This in turn leads to an accumulation of double-strand breaks in cells with a defective homologous repair (HR) machinery and eventually results in cell death. HR deficiency is most commonly caused by mutations in important DNA repair genes such as ensitive ,6,7.BRCA1/2, loss of PARP1 expression, or increased expression of ATP-binding cassette (ABC) transporters, effectively pumping the PARPi out of the cells. Another previously investigated mechanism involves tumors overexpressing the tyrosine kinase receptor MET, which is a common feature in many cancer forms [Nevertheless, many patients develop resistance to PARPi , and thier forms . High MEer forms , triple-er forms , and noner forms .Overcoming treatment resistance in an era of targeted treatments is an unmet clinical need. Several studies report that MET can phosphorylate PARP1, thereby preventing binding of the inhibitor and thus making cells PARPi-resistant ,14. The Another possible mechanism underlying the development of resistance and relapse is that tumors may harbor cancer stem cells capable of evading chemotherapy treatment . An incrSOX2 and POU5F1 (encoding OCT4) mRNA levels, an enrichment of ALDH-, CD133-, and CD117-positive cells, and a significant increase in sphere-forming capacity following PARPi treatment of ovarian cancer cell lines [Interestingly, connecting these two possible resistance mechanisms, overexpression of MET has been linked to increased levels of the stem cell markers ALDH1A3 and CD133 in breast cancer . Nozaki ll lines . Finallyll lines .BRCA1 or BRCA2 and in their counterparts with restored BRCA1/2 function to emulate platinum-resistant disease. The purpose was to explore if the capacity to activate a cancer stem cell network might be a clue to the ability of BRCA1/2-proficient cells to evade PARPi treatment.The aim of this study was to assess whether MET overexpression or co-expression of MET and SOX2 showed any prognostic potential in a consecutive and well-annotated contemporary cohort of advanced HGSC. Based on the links between MET and cancer stem cells in multiple tumor types and the potential role of MET in treatment resistance, we also wanted to investigate the role of stem cell factors in the proposed synergistic effect of METi and PARPi in HGSC cell lines with mutations in either Ethical approval for this study was granted by the Lund University ethics committee (Sweden), waiving the requirement for informed consent EPN 2014/717). A consecutive cohort of 156 HGSC cases, Cohort 1, was collected at the Department of Obstetrics and Gynecology at Skåne University Hospital (Sweden) between 2011 and 2015. All cases were reviewed by a gynecologic pathologist according to the World Health Organization Classification 2014  and stag14/717. ATMA sections, 3 µm in thickness, were stained with a primary antibody anti-c-Met  using the fully automated DISCOVERY ULTRA (Ventana). Antigen retrieval was performed using CC1  for 64 min at 95 °C, and the slides were incubated with ready-to-use primary antibody for 32 min at 37 °C. The TMA was scored using PathXL (Philips) by two independent examiners (MB and LT). In case of discordance, a consensus for each core was reached through joint reevaluation and, when needed, a trained gynecologic pathologist was consulted (AM). For each patient, a mean of the cores was calculated, and expression of MET was grouped as membranous staining in 0%, <5%, 5–9%, 10–49%, or 50–100% positive cells, with either weak or moderate intensity (representative images of intensity levels are shown in mRNA data from 489 HGSC patients included in the 2009 TCGA study  was retrBRCA1/2 mutations (germline or somatic), and those positive received olaparib treatment in line with clinical guidelines at the time. Pathological BRCA1 or BRCA2 mutations were found in 10/25 patients. Ethical approval for this cohort was granted by the Lund University ethics committee (EPN 2016/508). Written informed consent was obtained from patients receiving the olaparib treatment, while the consent requirement was waived for the remaining participants.An additional, partially overlapping cohort of 25 HGSC patients was used for global copy number analyses (referred to as Cohort 2). This cohort was collected between 2015 and 2016 and consists of patients presenting with relapsed high-grade serous ovarian, tubal, or peritoneal carcinomas who had at least partial response to second-line platinum treatment. Patients were screened for ® FFPE Assay Kit (Affymetrix) was used with 80 ng DNA as input. Segmentation, calculation of ploidy, normal cell contamination, and allele-specific copy number analyses were performed using ASCAT [For this cohort, DNA was extracted from FFPE tumor tissue using the Qiagen Allprep kit for FFPE tissue (Qiagen). Sections of the tissue were stained for validation of tumor content. SNP array analysis was performed at Eurofins Genomics Europe Genotyping A/S. The OncoScanng ASCAT   were cultured in RPMI-1640 HyClone with 10% FBS, 2 mM glutamine, 2 mM sodium pyruvate, and 1% penicillin–streptomycin . The UWB1.289 cell line is derived from an ovarian tumor of papillary serous histology with a BRCA1 mutation and defective DNA repair. UWB1.289+BRCA1 cells are from the same line, but in this version, a BRCA1-containing vector was stably transfected. UWB1.289 and UWB1.289+BRCA1 (ATCC) were cultured in 50% MEGM  and 50% RPMI-1640 HyClone (Nordic Biolabs). Media were supplemented with 3% FBS, and for UWB1.289+BRCA1, 200 ug/mL G-418  was additionally added. Mycoplasma testing and cell line authentication for all four lines was performed by GATC .Four HGSC cell lines were cultured. The PEO1 and PEO4 cell lines are derived from ascites from a patient with poorly differentiated serous adenocarcinoma. PEO1 was collected at first relapse when the tumor was considered platinum-sensitive, and PEO4 was collected after the second relapse, when the tumor no longer responded to platinum . A BRCA2w/v trichloroacetic acid , and air-dried. Cells were stained with 0.4% (m/v) sulforhodamine B (Sigma Aldrich), dissolved in 1% acetic acid for 30 min, and subsequently washed with 1% acetic acid. Plates were air-dried, and the dye was dissolved in 10 mM TRIS Base  before the absorbance was measured.Cells were seeded in 96-well plates, and single-drug treatment was performed with a range of concentrations for carboplatin, olaparib, and crizotinib, all purchased from Selleckchem. For combination treatment assays with olaparib and crizotinib, several doses for each drug were used, and the concentrations were chosen specifically for each cell line, ranging between approximately IC5 and IC60, as determined from the single-drug treatment growth assays. Cells were incubated with drugs for 6 days, fixed with 17% Copy number and mRNA expression data for the cell lines PEO1 and PEO4 were retrieved from CCLE , 21Q1 Public). Data had been log2-transformed using a pseudo-count of 1. The panel of 40 HR-associated genes described earlier  was used® (Bio-Rad), and intensity was normalized to stain-free blots.UWB1.289 and UWB1.289+BRCA1 cells were collected by scraping on ice, and Western blot analyses were performed using TGX stain-free gels (Bio-Rad Laboratories). Following blocking in 5% milk on a shaker at 4 °C overnight, membranes were incubated with primary antibodies against SOX2 , Nanog , OCT4 , RAD51 , BRCA1 , and Met  on a shaker overnight at 4 °C. The primary antibody HSP90 , used to ensure equal protein loading on gels, was applied to the membranes for 2 h at room temperature. The membranes were washed and incubated with HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (Thermo Fisher Scientific) for 1 h at room temperature. Detection was performed with Luminata Forte Western HRD Substrate  or SuperSignal West Femto Maximum Sensitivity Substrate , using Bio-Rad’s Stain-Free imaging technology . Quantification of Western blot results was performed using ImageLabt-test alternatively linear regression was used, as indicated in the Results section. For ordinal, non-parametric data  Wilcoxon rank–sum test was used, and for non-ordinal variables  Fisher’s exact test was used. To evaluate survival outcome, Cox regression was used both in a univariate model comparing MET-positive/MET-negative patients, and in a multivariate model where also age, stage, and residual tumor left after surgery were included. Age was used as a continuous variable, and stage and residual tumor as binary variables (III/IV and yes/no). Spearman rank correlation (rho) was used to evaluate co-expression of MET and SOX2 at different levels of expression. For Cox regression, when including an interaction term for MET and SOX2, MET-negative and SOX2-negative were marked as reference. All calculations, visualizations, and statistical analyses were performed using R statistical environment version 4.0.5 [To analyze the difference in the mean of continuous variables, Student’s on 4.0.5 .BRCA1/2 mutation status, and amount of tumor remaining after debulking surgery . MET-positive staining (defined as ≥5% cells with membrane staining) was found for 31 out of 130 patients. The groups of patients with MET-positive vs. MET-negative tumors were comparable in age,  surgery . Patientp = 0.62). When combining data for MET and SOX2 expression, we found that the subgroup of patients with tumors negative for SOX2 expression separated from those positive for SOX2. In the SOX2-negative subgroup, patients with MET-positive tumors had a significantly worse outcome than those negative for MET , indicating the significant impact of these markers on outcome. There was no significant difference in MET expression between primary and metastatic tissue .Kaplan–Meier analyses of OS  and PFS  revealed shorter survival for patients with MET-positive tumors compared to those with MET-negative tumors . SimilarMET mRNA expression alone and in combination with four closely connected transcription factors, described as stem cell markers  using a cut-off of z-score > 0 to define the expression of the individual markers. None of these markers were independently connected to OS .To further investigate the role of SOX2 and other stem cell markers in relation to MET expression, we retrieved a TCGA data set consisting of 489 patients, 461 of whom had both clinical and mRNA data available . We evaled to OS , but wheBRCA1/2-mutated and -wildtype samples, separately, are shown in BRCA1/2-mutant and -wildtype groups, but no significant difference was found . OS was longer for patients with a BRCA1/2 mutation, however, not to a significant degree , however, not to a significant degree (Student’s t-test p = 0.10).HRD scores were calculated in 25 HGSC tumors with available copy number data (Cohort 2). HRD scores in this selected cohort ranged from 26 to 85, with a median of 57. Clinico-pathological information is shown in BRCA1/2 status, HRD scores, and survival outcomes are displayed in SOX2 and HRD score . MET amplification and expression were rare in this cohort , a PARPi (olaparib), and a METi (crizotinib). The cell lines PEO1 and PEO4 were obtained from the same patient before and after platinum resistance was established. UWB1.289 and UWB1.289+BRCA1 originate from the same patient, and UWB1.289 is the parental cell line from which the UWB1.289+BRCA1 line was generated upon stable transfection with a plasmid containing a full-length functional BRCA2-deficient PEO1 and BRCA2-proficient PEO4 cells. By investigating the expression of HR-associated core genes, we found that these genes in general exhibited higher expression in the BRCA2-mutant PEO1 cell line compared to PEO4. For 20 genes, expression was higher in PEO1 cells, and for 6 genes, expression was higher in PEO4 cells. Copy numbers, however, were comparable between the cell lines. mRNA expression levels differed markedly between the cell lines for the stem cell factor POU5F1 (encoding OCT4) as well as the stemness-associated genes ALDH1A3 and PROM1 (encoding CD133), with higher levels in PEO1 than in PEO4 cells. Copy number and mRNA data for stem cell factors, as well as the crizotinib targets ALK, MET, and ROS1, DNA repair markers RAD51 and BRCA1/2, and genes involved in the MET signaling pathway, are displayed in Copy number and mRNA expression data for PEO1 and PEO4, available from the CCLE database, were examined for a panel of 40 HR-associated genes  as well BRCA1-proficient cell line were not available in the CCLE database. Results are displayed in BRCA1-proficient cell line. Nanog levels were below the level of detection by Western blot in both lines.Western blot analyses for the presence of MET, the stem cell factors SOX2, Nanog, and OCT4, as well as the HR markers RAD51 and BRCA1 were performed for the UWB1.289 and UWB1.289+BRCA1 cell lines, as mRNA data for the MET was discovered to be a potent oncogene in the 1990s when studies reported that when co-expressed with its ligand, hepatocyte growth factor (HGF), it promoted metastases in an animal model [MET did not have an effect on survival in the SOX2-negative subgroup; however, in the POU5F1-negative subgroup, we saw similar effects as in the TMA cohort. The SOX2 and OCT4 proteins interact closely by forming dimers with the ability to bind specifically to genes involved in cell fate and pluripotency [SOX2, POU5F1, Nanog, and KLF4, we observed that MET expression affected OS in patients whose tumors expressed none or only one of the factors. This indicates that these factors may complement each other and suggests that the tumor relies on other mechanisms for progression when an insufficient number of stem cell factors are present. Further studies are required to confirm these theories. In this context, Robinson et al. recently reported a stronger role for SOX2 compared to OCT4 or Nanog for tumor relapse potential following chemotherapy in ovarian cancer patients and that HGSC cell lines displayed greater chemoresistance and increased expression of SOX2, OCT4, and Nanog when grown in 3D conditions compared to 2D [al model . Overexpal model . The proal model . We set ipotency . Both coipotency ,38. The ed to 2D .BRCA1/2 mutations. This may be due to generally high HRD scores in this cohort, since all patients were platinum-sensitive and had at least a partial response to re-treatment with platinum. The HRD scores for the whole cohort were nonetheless comparable to the range of scores previously reported in HGSC [BRCA1/2 mutations had a longer OS, which could be expected as they received olaparib treatment and, as a group, also generally respond better to platinum treatment [To explore potential links between PARPi response, copy number aberrations, and cancer stem cell factors, we compared the copy number landscape and HRD scores in a cohort consisting of patients receiving either olaparib maintenance or surveillance (Cohort 2). We found, surprisingly, small differences between the groups with and without  in HGSC . Using t in HGSC , 84% of reatment .SOX2 was common, as has previously been reported in several cancer forms, including HGSC [MECOM, PIK3CA, ECT2, and PRKCI). Whether SOX2 should be considered a passenger or a driver gene is not fully determined. In this study, SOX2 protein levels appeared to correlate with SOX2 amplification, supporting the notion that it plays a role in cancer development and that overexpression of SOX2 to some extent is dependent on amplification.Amplification of ing HGSC . The comBRCA2 mutation, and PEO4 has a reversal mutation restoring BRCA2 function. This result contradicts earlier reports, which stated that the PEO1 line was more sensitive [BRCA2 function. PEO1 also expressed higher levels of several key stem cell factors, which could indicate a more stem cell-like phenotype. As cancer stem cells have been reported to express higher levels of drug transporter pumps, have a more intact DNA repair system, and are more prone to epithelial-to-mesenchymal transition (EMT) [Comparing growth inhibition in cell line pairs, we found that the post-treatment platinum-resistant PEO4 cells were more sensitive to olaparib treatment than PEO1 cells, although PEO1 cells harbor a ensitive ,42. We fon (EMT) , a higheon (EMT) , and appBRCA1-proficient line compared to the BRCA1-deficient line. This may indicate that the platinum and PARPi resistance acquired through the restoration of the BRCA1 function is connected to the activation of stem cell networks. Of note, the recently concluded PRIMA trial reported benefit in all patient groups receiving niraparib as first-line maintenance treatment, provided complete or partial response to upfront platinum therapy irrespective of the HRD status [In order to compare gene activity in the UWB1.289 and UWB1.289+BRCA1 cell lines, Western blot analyses were performed on selected proteins, as the UWB1.289+BRCA1 cell line was not included in the CCLE DepMap mRNA data set. The Western blot analyses revealed markedly higher protein expression of SOX2 in the D status , clearlySynergistic effects of olaparib and the c-MET inhibitor crizotinib have been reported in earlier studies, including ours ,16,17, bWe conclude that MET expression could be used as a prognostic tool in HGSC, but measures of stemness should be taken into consideration when further evaluating the mechanism/s underlying the driving effect of this receptor tyrosine kinase in cancer. Combining MET and PARP inhibitors did not increase cancer cell death in vitro in this study; nevertheless, MET inhibition could still be a viable treatment option. It should, however, be noted that patients with tumors expressing stem cell factors might not benefit from the treatment. Including stem cell marker screening in future clinical trials could help pinpoint the subgroup of patients eligible for METi treatment.


The incidence of depression among humans is growing worldwide, and so is the use of antidepressants. However, our fundamental understanding regarding the mechanisms by which these drugs function and their off-target effects against human sexuality remains poorly defined. The present study aimed to determine their differential toxicity on mouse spermatogenic cells and provide mechanistic data of cell-specific response to antidepressant and neuroleptic drug treatment. To directly test reprotoxicity, the spermatogenic cells (GC-1 spg and GC-2 spd cells) were incubated for 48 and 96 h with amitriptyline (hydrochloride) (AMI), escitalopram (ESC), fluoxetine (hydrochloride) (FLU), imipramine (hydrochloride) (IMI), mirtazapine (MIR), olanzapine (OLZ), reboxetine (mesylate) (REB), and venlafaxine (hydrochloride) (VEN), and several cellular and biochemical features were assessed. Obtained results reveal that all investigated substances showed considerable reprotoxic potency leading to micronuclei formation, which, in turn, resulted in upregulation of telomeric binding factor (TRF1/TRF2) protein expression. The TRF-based response was strictly dependent on p53/p21 signaling and was followed by irreversible G2/M cell cycle arrest and finally initiation of apoptotic cell death. In conclusion, our findings suggest that antidepressants promote a telomere-focused DNA damage response in germ cell lines, which broadens the established view of antidepressants’ and neuroleptic drugs’ toxicity and points to the need for further research in this topic with the use of in vivo models and human samples. According to the World Health Organization (WHO), depressive disorders are the greatest burden of disease in developed countries, affect millions of people, and are the leading cause of disability around the world . DepressPsychiatrists routinely use antidepressants to treat depression, making their application significant and growing worldwide . In addiThe exact mechanism of the effect of antidepressants on human sexual function is unknown, but most of them modulate the concentration of central neurotransmitters such as serotonin, norepinephrine, and dopamine. Serotonin inhibits the production of nitric oxide, which physiologically plays a key role in the sexual response, and its elevated levels impair sexual function. Furthermore, norepinephrine and dopamine are also involved in the cycle of sexual response related to desire and arousal. Many antidepressants can also suppress normal sexual function through cholinergic and alpha-1 adrenergic receptors ,16. ImpoThe purpose of this study was to determine the effect of selected antidepressants and neuroleptic drugs on mouse spermatogenic cells (GC-1 spg and GC-2 spd cell lines) and provide mechanistic data of cell-specific responses to antidepressant treatment. This preliminary study becomes an important aspect in terms of effectiveness for treating depression and will provide clinically relevant data associated with side effects of drugs and male fertility reduction.p < 0.05, p < 0.001). Interestingly, at the same time, the GC-2 spd cell line was characterized by a significant decrease in ATP level  (p < 0.001). The most pronounced decrease was noted for cells treated with MIR and VEN (both cell lines tested) after 96 h incubation (p < 0.001). For the rest of the drugs analyzed, a statistically significant decrease in the ATP was also observed after 96 h incubation. The exceptions were two drugs—ESC and OLZ (GC-1 spg)—where the ATP level remained unaffected  (GC-1 spg) and VEN (p < 0.01) (GC-2 spd). In turn, for ESC and REB, the S phase of the cycle was reduced (p < 0.05) (GC-2 spd only). Along with longer exposure to antidepressants, a significant reduction of the G0-G1 phase (p < 0.01–0.001) with a simultaneous extension of the S (p < 0.05) and G2/M phases (p < 0.05–0.01) (GC-1 spg) was observed for most experimental sets. A similar trend was not confirmed for the GC-2 spd cell line (p < 0.05–0.001). The second line, in general, also showed an increase in the synthesis level of these proteins after 48 and 96 h (p < 0.05–0.001) of incubation with drugs  caused a reduction in cell population in G0-G1 phase for FLU . The highest number was noted for ESC, FLU, and VEN in all experimental sets. Only MIR and in part REB drugs were characterized by the percentage of TUNEL + cells at the control level A,C. In aPersistent stress can lead to further damage to the nucleic acids. We noted a significant increase in micronuclei production after 48 h (except IMI) and 96 h incubation for both cell lines tested. Interestingly, after 96 h exposure, we observed a slight decrease in the amount of MN compared to 48 h, and this trend was observed for all experimental sets A–D. SimuNext, we assessed the interaction between nuclear mitotic apparatus protein (NuMa) and α-tubulin. We noted a decrease in NuMa/α-tubulin ratio intensity in most experimental sets of the GC-1 spg cells (48 and 96 h) except AMI and ESC (48 h) A,B. A siFinally, we have conclusively confirmed cell death resulting from exposure to antidepressants. We observed a significant decrease in cell viability after 48 h incubation with drugs for the GC-1 spg . The effect was even more pronounced after 96 h of exposure A,B. In t2+ reuptake, which leads to their overload and cell death  × 100%.The immunostaining was done accordingly to Mytych et al. 2017 . Brieflyp-value of <0.05 and considered as statistically significant . (*) indicates a comparison between non-treated and antidepressants-treated cells, and (^) indicates a comparison between the same drugs in different periods .All statistical analyses were carried out using Microsoft Excel and GraphPad Prism 6. The results are presented as the mean ± standard deviation of at least three independent experiments. The data were normalized separately for 48 h and 96 h experimental set-ups, with the control group being considered as 100%. The calculations were performed with a one-way analysis of variance and Dunnett’s posttest. The significance level was set at In conclusion, antidepressants are characterized by a delayed onset of therapeutic action, which requires long-term use. The use of this type of drug may be associated with side effects. Few reports also indicate that this may result in delayed various disorders of life functions, including reproductive functions and processes. Here, for the first time to our knowledge, we provide molecular evidence of possibly irreversible disorders in the course of spermatogenesis by germ cell damage.


Rhabdomyosarcomas (RMS) are soft tissue sarcomas predominant in the pediatric age range. Current standard multi-modal treatments can result in severe life-long side-effects across all subtypes and dismal outcomes in RMS patients classified as high-risk. There is an urgent need to find novel, more targeted therapies for these children. High expression of a number of histone demethylases is shown to support proliferation of RMS cells and here we direct our attention on KDM4B which has become a recent focus for drug discovery efforts. We show that KDM4B regulates cell cycle genes in the context of RMS cells and demonstrate that long-term silencing of KDM4B is functionally compensated for by another KDM4 family member, KDM4A. Upfront reduction of both KDM4A and KDM4B kills RMS cells demonstrating that therapeutics designed to target both histone demethylase family members is desirable to circumvent functional redundancy.CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including  Epigenetic marks on chromatin and the enzymes that modulate them are emerging as key mechanisms underlying cancer tumorigenesis and progression. Histone modifications are central to controlling chromatin architecture, gene transcription and DNA repair, and a number of proteins controlling the reading, writing and erasing of such marks are aberrant in cancer with targeted epigenetic modulators against them undergoing clinical trials .Histone demethylases (HDMs) are a family of epigenetic modulators responsible for the removal of methyl groups from specific residues on histone tails and have been shown to play a role in stem cell identity and differentiation ,3. The KKDM4 family members have also been shown to be genetically aberrant and overexpressed in certain malignancies ,8. MoreoCDK6 and CCNA2 via H3K9me3 at their promoters. However, after sustained silencing of KDM4B, we also show that recovery of cell growth is attributable to KDM4A recruitment to the promoters of CDK6 and CCNA2. Furthermore, upfront knockdown of both KDM4B and KDM4A led to cell apoptosis, not seen with silencing of either demethylase alone. Taken together, our results support therapeutic potential for RMS patients by KDM4 family inhibition.Rhabdomyosarcomas (RMS) are a type of soft tissue sarcoma that resemble developing skeletal muscle and are a major cause of cancer-related deaths in children and adolescents. Despite an overall increase in survival rate over the past few decades, drug resistance is a major factor in treatment failure . A numbe2.RMS cell lines RH30, RD, RMS-YM, RH4, JR-1, RMS-01, RH41, HFF-1 have been described previously . Cell liCell lines were authenticated using the Geneprint 10 kit according to manufacturer’s instructions  and subsequent fragment length analysis was carried out by Eurofins Genomics . The resulting short tandem repeat (STR) typing results were compared with public databases.For siRNA silencing experiments 3 siRNAs were used for each histone methyltransferase family member. siRNAs were synthesised by Sigma and RMS cell lines were transfected using Lipofectamine RNAiMAX  with siRNAs at a final concentration of 10 nM. The data was subsequently processed and analysed using python 3 using pandas, numpy, matplotlib, seaborn libraries B. For su® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) at 96 h. GI50s were calculated in GraphPad Prism 7  using the mean of six replicates. Cells for dose-response curves using were plated in 96-well plates and JIB-04  was added at increasing dose increments from 0 (DMSO (Sigma-Aldrich) only) to 10 µM and growth measured using using CellTiter 96Cells were fixed in 2% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100, blocked with 10% normal donkey serum with 1% BSA, and incubated with primary antibody O/N. Alexa Fluor 555-conjugated secondary antibody was used. Nuclei were stained with DAPI before fluorescence imaging . Antibodies listed in 2O2 at room temperature for 5 min. The sections were blocked with 4% BSA and incubated with KDM4B antibody at 4 °C overnight. Then, the samples were probed with secondary Ab , K5007, DAKO, Denmark), followed by incubation with peroxidase substrate solution  and counterstained with hematoxylin.The immunohistochemistry was performed on tissue microarrays containing samples from 219 RMS patients as well as normal tissues. Tissue microarrays were deparaffinized, hydrated through graded alcohol series and rinsed in water. After antigen retrieval by HIER, peroxide blocking was performed with 3% HThe immunoreactions were evaluated independently by two pathologists blinded to the clinicopathologic information. KDM4B immunostaining was evaluated based on the percentage of positively stained tumor cells. Additionally, the staining intensities were classified into the following four categories: 0 (no staining); 1 (weak staining); 2 (moderate staining), 3 (strong staining) in at least 10% of cells. Gene expression data described previously ,18 was a® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) and CyQuant Proliferation Assay kits  according to the manufacturer’s instructions. For cell cycle analysis cells were harvested after siRNA silencing, washed in 1XPBS fixed in 70% ethanol and stained for 30 min with propidium iodide (Invitrogen). Cells were then analyzed for to cell cycle distribution on an LSR II . Apoptosis assays were carried out after siRNA transfection using Caspase Glo assay (Promega). Cell proliferation assays were carried out using CellTiter 96RNA for RNA-sequencing was extracted 48 and 72 h post-transfection with either a non-targeting control siRNA or one of 2 KDM4B siRNAs (two replicates of each). RNA was sent to Oxford Gene Technology  for library preparation and sequencing. Gene expression data was obtained and analysed for differential gene expression between control siRNAs and KDM4B siRNAs.The diff files from the RNASeq were processed using R  using dplyr, ggplot2, gplots, RColorBrewer packages to manipulate the data and generate the scatterplots and heatmaps. The full data set was clustered using the R hclust hierarchical clustering function. Using this the data was split into 10 clusters for uploading to genego.com for GO analysis. Further analysis of the clusters was analysed in Python 3.Protein expression of KDM4 family members and cell cycle proteins was carried out by Western blotting. Protein from transfected cells was extracted using cell lysis buffer . Proteins were resolved on 3–8% Bis-Tris acrylamide gels and transferred onto PVDF membranes using the X-Cell II and iBlot systems according to manufacturer’s instructions (Invitrogen). Antibodies can be found in Chromatin immunoprecipitations (ChIP) were carried out using the SimpleChIP Enzymatic Chromatin IP kit in accordance with the manufacturer’s instructions . Eluted chromatin underwent qPCR analysis using primers specific to promoter regions of CDK6 and CCNA2. Primary antibodies can be found in Stable KDM4B and control shRNAs were packaged into virus in HEK293T cells as described previously . HEK293Tt-test (two-tailed) in GraphPad Prism 8.0.2 softwaree. Data is presented as the mean ± standard deviation. Significance is indicated as p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***), while NS indicates a non-significant p-value.Each examination was performed in at least triplicate. For in vitro experiments, statistical differences were analyzed using Welch’s 50 and compared these with known sensitive (MCF-7) and insensitive (HT-29) cell lines [As we had previously shown that a number of demethylase gene family members are overexpressed in RMS relative to normal skeletal muscle samples  we deterll lines . All RMSz-score of >−0.5 at either time point in each of the two cell lines. Overlaying the hits from the screen with those determined to be overexpressed in RMS patient samples, both fusion positive and negative  and alveolar RMS , at two time-points using three siRNAs for each HDM . Briefly 0.0001, ) identif 0.0001,  and the p < 0.0001, one-way analysis of variance) and a panel of normal tissues , with no statistical difference in expression between fusion gene positive and negative RMS  (p = 0.001) , all of which have long-term follow-up clinical information available. 40% of RMS were positive for KDM4B staining (>10% cells stained), comparing to negative staining in normal muscle (< 0.001) , consist< 0.001) . Based o= 0.001) , consistA role for KDM4B in cell proliferation was assessed after RNAi-mediated silencing of KDM4B using KDM4B-specific siRNAs in 6 RMS cell lines representing both ERMS and ARMS and compared with HFF-1 cells. KDM4B silencing resulted in a reduction in cell proliferation in both ARMS and ERMS cell lines of up to 70% by 96 h post transfection . To confTo determine which genes may be responsible for the phenotypic effects seen in ARMS cell lines, we investigated the transcriptional effect of silencing KDM4B in the ARMS line RH30 by RNA-sequencing at 48 and 72 h post-transfection. Gene Set Enrichment Analysis (GSEA) revealed both overlapping and distinct genes that were down-regulated at 48 h and 72 h post-transfection –D. Gene Of the genes that were down-regulated at either 48 h and/or 72 h after KDM4B RNAi we noted that a number of these are known to be involved in cell cycle checkpoint control at different phases of the cell cycle, including CDK6, a G1-S checkpoint gene, and CCNA2, a G2-M checkpoint gene. To confirm that KDM4B is regulating these genes in RMS we performed KDM4B silencing in four RMS cell lines and confirmed a reduction in CDK6 and Cyclin A2 (CCNA2) expression by Western blot in all cell lines . In the p < 0.03, Day 17, p = NS, To determine the long-term effects of sustained KDM4B-silencing in RMS cells we tested 6 different shRNAs directed against KDM4B . We measAs functional redundancy has been postulated to exist between KDM4 sub-family members ,3, we soDespite a lack of upregulation in expression of other KDM4 family members, there remains the possibility of functional redundancy in regulating genomic targets of KDM4B in its absence. To test this hypothesis and determine whether the lack of reduction in expression of KDM4B-controlled cell cycle genes could be compensated for by the presence of KDM4A on these promoters we analyzed KDM4A binding on the promoter of CDK6 and CCNA2. In RH30 cells transduced with control shRNA, KDM4A does not bind to these promoters. However, after KDM4B silencing by shRNA, we observed an enrichment of KDM4A at these promoters, suggesting that KDM4A is now regulating expression of CDK6 C and CCNAs sustained silencing of KDM4B led to an increase in KDM4A at the CDK6 promoter we hypothesized that simultaneous targeting of both KDM4A and KDM4B may result in a stronger phenotype and thus mitigate the effects of stable silencing of KDM4B alone. siRNA-mediated dual targeting of KDM4A and KDM4B lead to a significantly greater decrease in RMS cell proliferation than with either KDM4A or KDM4B alone A,C. FurtIn this study, we demonstrate for the first time the role of KDM4B in controlling cell proliferation and cell cycle progression in RMS. Moreover, in this context, we also show the existence of functional redundancy between KDM4B and KDM4A in cell cycle regulation. Building on our previous study showing that HDMs are highly expressed in RMS compared to normal tissues, here we demonstrate that a number of HDMs affect RMS cell growth and show that silencing of KDM4 family members results in a reduction in cell proliferation in RMS cell lines including the H3K9me3 demethylase KDM4B. CDK6 in bladder and lung cancer cells [CDK6 in RMS and may therefore be a more widespread driver of proliferation in tumors. This phenotype is enhanced through the associated expression of several other cell cycle checkpoint genes known to be expressed at different phases of the cell cycle. Our RNA-sequencing data revealed that cell cycle genes, including CDK6, CCNA2, SKP2 and MYC were all down regulated after KDM4B silencing. SKP2 has been shown to be highly expressed in RMS and is a downstream target of the PAX3-FOXO1 fusion protein [SKP2 in RMS via H3K9me3 regulation. Interestingly, silencing of KDM4B in ERMS cell lines resulted in cell cycle arrest, compared to ARMS cell lines in which silencing of KDM4B results in a slowing down cell cycle progression. KDM4B has been shown to regulate different genes dependent on cell context . As ARMiewed in  it is liHere we show that KDM4B plays a role in the proliferative capacity of RMS cells. Indeed, in stem cells expression of KDM4 proteins is required for maintaining the undifferentiated, proliferative state and regulating differentiation ,3,5,6. RRNA-sequencing following KDM4B silencing also showed that the majority of genes that changed were down-regulated, consistent with transcriptional activation via H3K9me3 demethylation by KDM4B. However, around 25% of genes were upregulated after KDM4B silencing, suggesting that these genes are regulated by the H3K36me3, a hallmark of active transcription, implicating a demethylating, repressive role of KDM4B on these genes. Mutations in histone H3 lysine 36 to methionine (H3K36M) have been shown to prevent differentiation in mesenchymal progenitor cells (MPCs) and have been linked to sarcomagenesis in mouse models . SilenciAlthough short-term silencing of KDM4B by siRNA in RMS cells led to a reduction in proliferation, long-term stable silencing by shRNA did not. This is in contrast to reports in neuroblastoma, where stable silencing of KDM4B led to a reduction in cell proliferation and a concomitant induction of differentiation in these cells . In RMS Over the past few decades drug discovery efforts have commonly sought a single molecule that selectively targets a single protein. However, functional redundancy between protein family members and/or signaling pathways have been shown to be potential mechanisms of resistance to therapies in the treatment of cancer ,29, and There has been a recent drive to find KDM4 inhibitors, although very few of these studies have been able to derive selective compounds without poly-pharmacology with other KDM families, largely due to the fact that they are either cofactor mimics or substrate competitive, both of which are often shared across histone demethylase families. However, some selectivity has been achieved using cyclic peptides, which even allow for some selectivity over subfamily members . Based oOverall, the data we have presented highlights the importance of understanding the underlying functional redundancy that may exist between histone demethylase family members in order to effectively target these epigenetic proteins for the effective treatment of cancer. Our discovery that KDM4A is able to compensate for down-regulated KDM4B by assuming the latter’s H3K9me3 demethylating role to drive transcription of KDM4B-regulated genes highlights the potential for sub-family targeting of these proteins rather than striving for single-target therapies. KDM4A itself has been associated with mechanisms that may lead to resistance , thus itFurthermore, our data show that effectively targeting both KDM4A and KDM4B together leads to apoptosis in RMS cells and thus suggests that effective dual targeting of these proteins could lead to a more effective therapy, whilst maintaining the benefits of more targeted therapies with reduced toxicities. A number of histone demethylases are highly expressed in rhabdomyosarcoma and impact on cell proliferation, including KDM4B. We determine that KDM4B regulates the expression of cell cycle genes in rhabdomyosarcoma. However, after clonal selection of KDM4B-silenced cells, KDM4A is able to functionally compensate for the regulatory effects of KDM4B on the promoters of the cell cycle genes CDK4 and CCNA2. We conclude that, in order to mitigate for this functional redundancy and enhance therapeutic potential for rhabdomyosarcoma patients, a dual targeting approach to pharmacological inhibition of these histone modifying enzymes will be necessary.


Pyrazoles are an interesting class of compounds showing potent anticancer activities. Our previous studies have demonstrated the potent anticancer activity of pyrazole analogues. Therefore, we focused on developing anticancer agents through structure optimization of the pyrazolyl lead molecule.  The pyrazole derivatives were prepared by the appropriate synthetic protocols. The antiproliferative activities were evaluated using a sulforhodamine B assay against three cancer cell lines. In vitro and in silico molecular docking studies employing xanthine oxidase were used to explore the mechanism by which pyrazole derivatives exert anticancer effects. 50 4.2 μM), with a potent xanthine oxidase inhibitory activity (IC50 0.83 μM).  One of the pyrazole derivatives demonstrated the greatest promise as an anticancer agent against the human colon cancer cell line (IC In summary, our findings suggest that these pyrazolyl analogues containing a pyridine nucleus could serve as a promising lead molecule in the development of novel anticancer agents. Cancer is one of the four major types of noncommunicable diseases, along with cardiovascular disease, diabetes, and chronic respiratory diseases . It is a50 value in the range of 0.2–3.4 μM  × 100, where Ac indicates the absorbance of the control sample and As is the absorbance of the treated sample. Readings were taken in triplicate and represented as IC50 ± SD. IC50 values were calculated using GraphPad Prism 8 software.These compounds were further evaluated for their XO inhibitory potential by measuring the formation of uric acid concentration spectrophotometrically at 292 nm using a method previously described . Solutiohttp://www.hyper.com). AutoDock tools (ADT) version 1.5.6 (http://www.autodock.scrips.edu) was used to prepare the molecular docking. The active site was considered as a rigid molecule, while the ligands were treated as being flexible. Using default parameters, grid-based docking studies were carried out and docking was performed on all compounds using the standard ligand oxipurinol. The best binding conformation was selected from the docking log (.dlg) file for each ligand, and further interaction analysis was performed using PyMOL and Discovery Studio Visualizer 4.0.Molecular docking studies were performed using AutoDock v. 4.2.2 to identify appropriate binding modes and conformation of the ligand molecules. The crystal structure of xanthine dehydrogenase  was retrieved from the RCSB Protein Data Bank as a PDB format . The str50 0.2–3.4 μM) as a potential anticancer lead molecule, we focused our attention on optimizing the anticancer potential of arylhydrazono-pyrazole derivatives with different substitutions at the phenyl ring , hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116), by the sulforhodamine B (SRB) assay. The SRB assay is the “gold-standard” assay and is extensively used in the high-throughput screening program at the National Cancer Institute (NCI), USA. The SRB uses an aminoxanthene dye, which readily stains the cells through binding with the basic amino acids of cellular proteins. The SRB assay estimates the dye released from the stained cells after washing as stoichiometric proportions of cellular mass. In 50) values. The IC50 values were determined by interpolation from dose-response curves. From the results, it was observed that most of the newly synthesized pyrazoles showed excellent to moderate antiproliferative activity against the three cell lines, especially against colorectal carcinoma (HCT-116). Compound 1 effectively inhibited cell growth at IC50 values of 17.8 ± 0.5, 4.4 ± 0.4, and 4.2 ± 0.2 μM against the breast cancer (MCF-7), hepatocellular carcinoma (HepG2), and colorectal carcinoma (HCT-116) cell lines, respectively. The anticancer activity of compound 1 was comparable to the standard drug doxorubicin against the HepG2 (3.9 ± 0.06) and HCT-116 (4.4 ± 0.04) cell lines, but 3.7 times less potent in the MCF-7 (4.7 ± 0.08) cell lines. Compound 2 was found to be moderately effective, with IC50 values of 94.2 ± 0.3, 34.6 ± 2.6, and 17.3 ± 0.5 μM against the MCF-7, HepG2, and HCT-116 cell lines, respectively. Our previous studies have shown that unsubstituted phenyl derivative of aryldiazenyl pyrazole had a poor antiproliferative activity (IC50 > 100 μM) against all three cell lines [Based on the encouraging results of 1-isonicotinoyl-3-methyl-4-[2-(4-nitrophenyl)hydrazono]-2-pyrazolin-5-one  exhibited significant XO inhibition, 18.03 times more potent than the standard drug allopurinol (IC50 14.97 ± 1.61 μM) (2 was equipotent (IC50 14.50 ± 2.25 μM) to allopurinol. These results confirm the XO inhibitory action of these compounds and suggest the possible influence of XO inhibition in the anticancer activity of pyrazoles. However, further kinetic studies are required to establish a more detailed mode of enzyme inhibition of these compounds.Pyrazolyl analogues have been reported as promising anticancer agents acting through xanthine oxidase (XO) inhibition . The pro1.61 μM) , whereas4+) of the molybdopterin cofactor, which is essential for the mechanism-based inhibition. Furthermore, it forms hydrogen bonds with Glu802, Arg880, and Glu1261 in the active site of the enzyme  using AutoDock. Both the enzymes XO and XDH are interconvertible forms of the same enzyme, known as xanthine oxidoreductase (XOR), with only slight differences in the FAD binding domain. The crystal structure of bovine milk XDH bound with oxipurinol was used in the current study and retrieved from the raw PDB structure 3BDJ, which has 90% similarity to the human liver enzyme . Oxipurie enzyme . These be enzyme . The keye enzyme . The res1 and 2 were prepared by using the method described earlier [50 > 100 μM), and compound 1 was about >22 folds more active than its unsubstituted derivative in the HepG2 and HCT-116 cell lines. These findings suggested that the electron withdrawing groups at the phenyl ring might be important for high potency. Furthermore, HCT-116 was the most sensitive cell line against these pyrazolyl analogues.After obtaining interesting results with arylhydrazono-pyrazole analogues, different substitutions were made at the phenyl ring for optimizing the template. The analogues  earlier . Their a1 emerged as a promising anticancer agent effective against the colorectal carcinoma cell lines (IC50 4.2 μM) acting through xanthine oxidase inhibition (IC50 0.83 μM). Results of in vitro and in silico xanthine oxidase inhibitory activities revealed that the anticancer activity of these pyrazolyl analogues might be due to XO inhibition.In conclusion, compound