Colony-forming units optimization and human papillomavirus detection in umbilical cord blood.

INTRODUCTION: Analysis of several parameters is required for adequate quality control in umbilical cord blood units (UCBU) when used for therapeutic purposes. OBJECTIVE: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. METHODS: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the sequence-specific priming technique. RESULTS: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY/11 and GP5/GP6+. CONCLUSIONS: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

INTRODUCCIÓN: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. OBJETIVO: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. MÉTODOS: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. RESULTADOS: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. CONCLUSIONES: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Thawing methods do not affect cell viability of CD45+ and CD34+ cells, but long-term cryopreservation of umbilical cord blood units generally decreases cell viability.

INTRODUCTION: Umbilical cord blood units (UCBUs) are collected and cryopreserved in biobanks for a future transplant. Hematopoietic stem cells and hematopoietic progenitor cells (HSC/HPC) present in UCB can be damaged due to factors such as the cryopreservation process, the thawing process, and prolonged storage time. METHODS: UCBUs (n = 27) were obtained from the Biobank of the National Center of Blood Transfusion (NCBT) from Mexico. They contained three attached segments of UCBU, including 1.0-2.3 × 106 CD34+ cells prior to cryopreservation and were stored during the period from 2003 to 2015. Each UCB segment was thawed with three different methods and CD34 cells, CD45 cells, and 7-AAD were identified by flow cytometry. Furthermore, we carried out CFU assays, and trypan blue staining. RESULTS: Viable CD45+ (vCD45+) cells, vCD34+ cells, CFU, and percentage of E-Clone were not statistically significant among three different thawing methods. The number of vCD45+ and vCD34+ cells diminished in the three thawing methods compared with the same cells prior to their cryopreservation (p < 0.0001). vCD45+ and vCD34+ cells diminished in the ≥10-year cryopreservation group (p < 0.001). In addition, percentage of recovery of vCD45+ and vCD34+ cells diminished in this same group (p = 0.013 and p < 0.0001, respectively). CONCLUSION: The thawing methods did not affect either cell viability (vCD45+ and vCD34+ cells) or pluripotency (CFU, percentage of E-Clone) in attached segments of UCBUs. The ≥10-year cryopreservation time in attached segments of UCBUs alters the number of vCD45+ and vCD34+ cells; however, it does not affect their pluripotency.

MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

Cell death induction by mycelium extracts from Pleurotus spp. on cervical cancer cell lines.

Mushrooms have health benefits, including anti-tumoral properties. We evaluated the cytotoxic and cell death induction effects of water-soluble extracts of Pleurotus ostreatus and Pleurotus eryngii mycelium in the cervical cancer cell lines HeLa (HVP18+) and SiHa (HVP16+) as well as the non-tumoral cell line HaCaT. Both Pleurotus extracts presented similar protein patterns from 190 to 10 kDa and displayed protease activity on a gelatine substrate. The mycelium extracts of both Pleurotus strains induced a dose-dependent cytotoxic effect on HPV+ cells IC50 65 µg), whereas HaCaT cells were less susceptible (IC50 90 µg). The cytotoxic effect at the IC50 concentration was not associated with apoptosis, the activation of Caspases-3/7 was not significantive; only P. eryngii induced a moderate (1.2-fold) increase in SiHa cells. Pleurotus extracts induced autophagy, mainly in SiHa cells (4.3-fold). Neither extracts induced changes in p53 protein expression, suggesting that the cytotoxic effect could be due to p53-independent pathways.

Overexpression of p53 protein is a marker of poor prognosis in Mexican women with breast cancer.

Breast cancer (BC) is a disease with different clinical, histological and molecular characteristics, frequently presenting mutated tumour-suppressing genes and oncogenes. P53 is a known tumour suppressor that is often mutated in BC; several mutations in p53 inhibit its role as a transcriptional repressor of several oncogenes. Topoisomerase 2α (TOP2α) is a gene target of p53, and it is also a known target for anthracyclines. The aim of the present study, was to analyse the genetic alterations of p53 and TOP2α genes and their levels of protein expression, as well as their association with survival in Mexican women with BC. A total of 102 biopsies were collected (tumour and adjacent tissues) from patients with BC. To identify point mutations and deletions in the p53 gene, the Sanger sequencing method was carried out. Deletions or amplifications for TOP2α gene were determined using quantitative polymerase chain reaction (qPCR). In addition, the expression of the TOP2α and p53 proteins was evaluated by western blotting. Furthermore, p53 protein expression was analysed by proximity ligation assay (PLA)-qPCR. Only 28.5% of the patients were found to have triple-negative breast cancer (TNBC); the average age at the time of diagnosis of these patients was 50 years, and Scarff-Bloom-Richardson (SBR) histological grade III (p=0.0089). No differences in point mutations or deletions in p53, and deletions or amplifications as well as protein expression level of TOP2α were observed between patients with TNBC and non-TNBC patients. However, patients with TNBC showed p53 protein overexpression as determined by PLA-qPCR and western blotting (p<0.0001). Furthermore, we found an association between TOP2α amplification and overexpression of its protein in patients with TNBC (p<0.0001). Concerning p53, overexpression resulted in a lower survival in patients with BC.

Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.