The purpose of this study was to determine the correlation between expression of HPV16 E6 p53 and p21 proteins and the physical state of HPV16 in cervical cytologies without squamous intraepithelial lesions (Non-SIL) and with low grade squamous intraepithelial lesions (LSIL) both with HPV16 infection. (SIL) of the cervix. Based on cytopathologic characteristics these lesions are divided into low grade (LSIL) and high grade (HSIL) and invasive cervical cancer [1]. Cervical cancer is usually a multistep process that slowly develops upon persistent contamination with oncogenic types of human papillomavirus (HPV) [2 3 In Mexico HPV16 is the most frequent genotype found in cervical cancer [4]. Moreover HPV16 is one of the most frequent genotypes found in LSIL HSIL and in women without SIL [5]. HPV16 genome encodes two oncoproteins E6 and E7. Both proteins are able to cause transformation of the host cell [6]. The E6 protein binds to p53 tumor suppressor and cause its degradation by 26S proteasome resulting in its inactivation and the impairment of p53-induced cellular apoptosis [7]. Expression of E6 has been proposed as a useful diagnostic and/or prognostic marker in cervical carcinogenesis [8] although presently PLX-4720 there are contradictory results [9]. p53 protein is known as the guardian of the genome and plays an important role in cellular response to genotoxic stress [10]. This protein acts as a tumor suppression by a variety of mechanisms including cell cycle arrest induction of apoptosis and cellular senescence PLX-4720 [11]. Previous studies have evaluated p53 protein expression in cervical intraepithelial lesions and in invasive carcinomas however the results are contradictory [12-15] making it difficult to establish whether p53 expression is a good PLX-4720 biomarker in cervical carcinogenesis. The p21 protein is member of the Cip/Kip family and PLX-4720 is responsible for cell cycle control blocking the transition from G1-phase to S-phase. The p21 gene is usually regulated through two different pathways a p53-dependent pathway and a p53-impartial way through platelet-derived fibroblast and epidermal growth factors [16 17 Reduced expression of p21 protein by immunohistochemistry has been reported in invasive squamous cell carcinoma [18] and it has been suggested as a biomarker. The HPV16 genome can be found in the host cell in episomal integrated or mixed forms. HPV16 integration into the host genome results in increased levels of E6 and E7 proteins and this event is considered a critical late-event in cervical carcinogenesis. The prevalence of episomal and integrated forms of HPV16 genome in cervical SIL varies with severity of disease [19 20 In general the integration of HPV16 genome is considered a late event in cervical carcinogenesis [21]. Several studies have suggested that this immunocytochemical or immunohistochemical detection of p16 [9] p53 p21 [22] cyclin A cyclin E [23] Ki-67 [24] telomerase [25] E6 [9] and the detection of physical state of HPV16 by ISH [23] in smears or cervical samples may provide useful diagnostic and prognostic information. The aim of this study was to determine the correlation between expression of E6 HPV16 p53 and p21 and the physical state of HPV16 in PLX-4720 cervical cytologies without squamous intraepithelial lesions (Non-SIL) and with low grade PLX-4720 squamous intraepithelial lesions (LSIL) both with contamination by HPV16 to identify possible biomarkers of early cervical lesion. Materials and methods Subjects and specimen collection 101 liquid-based cervical cytology samples were collected from women residents in the State of Guerrero in Southern Mexico. The study population consisted of 50 women diagnosed with Non-SIL and 51 diagnosed with LSIL all positive to HPV16 by PCR. Exo-endocervical exfoliated cell samples were collected by sampling the ectocervix with an Ayre spatula and endocervix with a cytobrush. Immediately after sample collection smears were prepared for cytomorphological examination through conventional Papanicolaou staining. The remaining cellular content was preserved in liquid base liquid-PREPTM (LPT) and used for immunocytochemistry and ISH. A second sample was collected for DNA extraction. All samples Pap smears were evaluated PCK1 by an experienced cytopathologist and were classified according to the Bethesda System [26]. All patients signed an informed consent and filled a questionnaire to obtain demographic data and information about gynecological risk factors. This project was approved by the Bioethics Committee of the Autonomous University of Guerrero Mexico and all procedures where in accordance with the ethical guidelines of the 2008 Helsinki Declaration. HPV detection and genotyping Genomic DNA was extracted from cervical cells by the phenol chloroform method.