Individual-oocyte transcriptomic analysis shows that genotoxic chemotherapy depletes human primordial follicle reserve in vivo by triggering proapoptotic pathways without growth activation.

Gonadotoxic chemotherapeutics, such as cyclophosphamide, can cause early menopause and infertility in women. Earlier histological studies showed ovarian reserve depletion via severe DNA damage and apoptosis, but others suggested activation of PI3K/PTEN/Akt pathway and follicle 'burn-out' as a cause. Using a human ovarian xenograft model, we performed single-cell RNA-sequencing on laser-captured individual primordial follicle oocytes 12 h after a single cyclophosphamide injection to determine the mechanisms of acute follicle loss after gonadotoxic chemotherapy. RNA-sequencing showed 190 differentially expressed genes between the cyclophosphamide- and vehicle-exposed oocytes. Ingenuity Pathway Analysis predicted a significant decrease in the expression of anti-apoptotic pro-Akt PECAM1 (p = 2.13E-09), IKBKE (p = 0.0001), and ANGPT1 (p = 0.003), and reduced activation of PI3K/PTEN/Akt after cyclophosphamide. The qRT-PCR and immunostaining confirmed that in primordial follicle oocytes, cyclophosphamide did not change the expressions of Akt (p = 0.9), rpS6 (p = 0.3), Foxo3a (p = 0.12) and anti-apoptotic Bcl2 (p = 0.17), nor affect their phosphorylation status. There was significantly increased DNA damage by γH2AX (p = 0.0002) and apoptosis by active-caspase-3 (p = 0.0001) staining in the primordial follicles and no change in the growing follicles 12 h after chemotherapy. These data support that the mechanism of acute follicle loss by cyclophosphamide is via apoptosis, rather than growth activation of primordial follicle oocytes in the human ovary.

Interferon-alpha elicits downregulation of human papillomavirus 18 mRNA in HeLa cells by selective repression of endogenous viral transcription.

We have reported that interferon-alpha inhibits HPV-18 mRNA in HeLa cells. Here we examine mechanisms by which IFN could modulate HPV expression. In northern blot experiments, we observed that interferon-alpha 2b treatment reduced HPV-18 mRNA levels in a time- and dose-dependent manner, with a maximal effect achieved at 48 h. Simultaneously, induction of 2-5A synthetase mRNA was verified as indicative of IFN action. The IFN regulatory effect on HPV-18 mRNA at 48 h required de novo protein synthesis. We performed run-on experiments to determine whether the IFN regulatory effect was at the transcriptional level. HPV-18 endogenous transcription was repressed using 200 and 1000 IU/ml. Interferon treatment did not affect HPV-18 mRNA stability, at least under our experimental conditions. To verify whether HPV-18 enhancer sequences were involved in the interferon effect, we transfected a construct containing the chloramphenicol acetyltransferase driven by the HPV-18 upstream regulatory region. The enzyme activity was unmodified on human keratinocytes and HeLa cells by interferon exposition. Our data demonstrate that interferon-alpha downregulates HPV-18 mRNA levels on HeLa cells by repressing nascent viral transcripts, possibly through regulatory cellular flanking regions.

Modulation of human papillomavirus type 16 mRNA in cervical invasive carcinoma patients by interferon-alpha therapy.

Mechanisms by which interferon produces papilloma regression remain largely unknown. We analyzed biopsies from three cervical invasive carcinoma patients treated with interferon-alpha (IFN-alpha) administered both topically and i.m. for 15 days. All specimens contained human papillomavirus (HPV-16) DNA as determined by polymerase chain reaction using specific HPV-16 E7 primers. Interestingly, in two patients. HPV-16 mRNA expression was reduced (44% and 67%, respectively) after IFN treatment. Upregulation of 2-5 A synthetase and PKR mRNA levels were indicative of the IFN effect. A larger study should be initiated to see whether IFN-alpha modulates the HPV-16 mRNA levels in tumor biopsies from cervical carcinoma patients.

Detection and typing of human papillomavirus DNA in benign and malignant tumours of laryngeal epithelium.

The role of human papillomaviruses (HPV) in laryngeal squamous cell carcinoma has not yet been established. Thirty-three cases of laryngeal squamous cell carcinoma were analysed for the presence of HPV DNA and compared with 25 cases of normal larynx and 29 cases of laryngeal squamous papilloma in their positivity index. The presence of HPV DNA was analysed by using L1 consensus primers and also by primers specific for the E7 gene of HPV types 16 and 18. Four normal laryngeal samples (16%) were positive for HPV DNA against the 24 samples (82%) (p < 0.001) found for laryngeal papilloma and 16 (48.5%) (p < 0.05) found for laryngeal squamous cell carcinoma. HPV 16 was the type most frequently found in laryngeal carcinoma samples. Our results support an etiologic role for this type of HPV in the pathogenesis of laryngeal carcinoma.

[Genetic expression of nerve growth factor in the central nervous system. Evaluation of an experimental model for Alzheimer's dementia]. / Expresión genética del factor de crecimiento neural en el sistema nervioso central. Evaluación en un modelo experimental de demencia de Alzheimer.

INTRODUCTION AND OBJECTIVE: Several authors have suggested that loss of neuronal trophic support may be an important element in the physiopathology of degenerative conditions of the central nervous system such as Alzheimer's dementia, Parkinson's disease or amyotrophic lateral sclerosis amongst others. In the light of present knowledge, the survival of cholinergic populations of the anterior basal cerebrum, closely involved with cognitive processes of memory and learning, is associated with adequate function of the neural growth factor (NGF). These populations are markedly damaged in Alzheimer's disease, and this has been correlated with the progressive loss of memory and intellectual involvement seen in this disorder. The model used in this study was based on section of the septohippocampal connecting pathways, so that transport of regulatory impulses from the hippocampus to the medial septum was interrupted. This has lethal results for the cholinergic neurons of the latter. We have developed a study designed to characterize the expression of the gene of NGF in different regions of the brain, involved in cholinergic neurotransmission in healthy and in damaged tissue. MATERIAL AND METHODS: We used a molecular hybridization technique with a cDNA catheter complementary to the radio-isotope marked NGF human gene. RESULTS AND CONCLUSIONS: The highest levels of expression were found in the healthy cortex and hippocampus. The reduction in the levels of mRNA of NGF in the damaged hippocampus supports the current thesis which considers synaptic activity to be a major regulator of the synthesis of this molecule in the brain.