BMC Cancer 2005, 5:77 http://www.biomedcentral.com/1471-2407/5/77 Background Uterine cervix carcinoma (UCC) represents the second cause of death among the female population worldwide. The fact that more than 99% of all the cervical invasive tumors are positive for infection with high risk human papillomavirus (HPV) suggests that this is one of the most important factors for the development of this neoplasm [1,2]. These viruses can induce cellular transformation by several mechanisms; the viral oncoproteins E6 and E7 can interact with cellular proteins involved in important cellu- lar functions, such as tumor suppression, apoptosis, cell cycle control, genomic instability, transcriptional regula- tion and immune evasion [3]. The induction of genomic instability by HPV seems to be particularly important for the establishment and develop- ment of an invasive tumor [4,5] since this increased genomic plasticity would generate cellular clones with enhanced transforming and invasive potential [6]. Metaphase comparative genomic hybridization (mCGH) has been applied to study different stages of this tumor [4,7-19], detecting specific patterns of chromosomal imbalances that arises very early during the development of cervical carcinoma, suggesting that the gain of chromo- some 3q is one of the most important genetic alteration that defines the transition from a pre-malignant lesion to an invasive carcinoma [4]. Some of these imbalances have been related to specific clinical behaviors, such as the pres- ence of lymph node metastases [9]. However, given the spatial resolution of mCGH [20], little is known about the identity of specific genes that might be the targets of regional chromosomal imbalances. Matrix-based CGH or array CGH overcomes this problem increasing the sensi- tivity for the detection of DNA copy number changes at specific loci, through the use of well defined genomic DNA fragments whose mapping location is known, arrayed onto a solid surface [21-23], thereby achieving a resolution of copy number imbalances up to the single gene level. In order to refine the patterns of chromosomal imbal- ances present in cervical carcinoma, and trying to identify specific genes that might be targets of copy number changes in this tumor, we applied microarray CGH on 20 uterine cervix-derived samples (three pre-malignant lesions, 10 invasive tumors and seven UCC derived cell lines) to detect DNA copy number changes at the single gene level. Methods Cervical tissues All described procedures have been evaluated and approved by the local committee of ethics of the Mexican Institute of Social Security (IMSS), and all samples were taken after informed consent from the patients. The pre- malignant lesions and the invasive tumors were collected by colposcopy-directed biopsies at the Gynecology Department of the Hospital General de México, Mexico City. The biopsies were divided in three sections. The cen- tral part was used for genomic DNA extraction using the Wizard Genomic kit (Promega, Madison, WI, USA), and the extremes were fixed with 70% ethanol overnight and paraffin embedded. Hematoxilin-eosin stained sections from these biopsies were analyzed in order to confirm the presence of at least 70% tumoral cells in the samples. Cell lines The cell lines included in this study were: CasKi, SiHa, both positive for HPV16, and HeLa (HPV18) The CaLo and ViBo cell lines were established from stage IIB inva- sive tumors, while INBL and RoVa from a stage IVA tumor. These cells are HPV18 positive and were established from tumor explants at the laboratory of Cell differentiation and Cancer of the National University of Mexico [24]. The chromosomal CGH profiles of CaLo, ViBo, INBL and RoVa have been published recently [19]. HPV detection and typing HPV detection was carried out by PCR using the consen- sus primers MY09 and MY11 for the L1 region of the viral genome. After a 5 min. denaturation at 94°C, 100 ng of DNA were subjected to 35 amplification cycles with the following parameters: 94°C for 1 min., 55°C for 2 min. and 73°C for 3 min., with a final extension step of 7 min. at 72°C. The amplicon was labeled using the Big Dye sequencing kit and sequenced on an ABI371 sequencer (Applied Biosystems, Foster City, CA, USA). BLAST http:/ /www.ncbi.nlm.nih.gov/BLAST/ sequence comparison was used in order to define the viral type. Microarray CGH Microarray CGH was performed using the GenoSensor Array 300 system, following the manufacturer's instruc- tions (ABBOT-Vysis, Downers Grove, IL, USA). Each array contains 861 spots, representing 287 chromosomal regions that are commonly altered in human cancer, such as telomeres, regions involved in microdeletions, onco- genes, and tumor suppressor genes. Briefly, 100 ng of genomic DNA were labeled by a random primer reaction during two hours. Tumor DNA was labeled with Cy3 and the normal female reference DNA with Cy5. After the labeling reaction, the probes were digested with DNAse at 15°C for one hr., followed by two ethanol-purifications; finally the probe size was checked by gel electrophoresis. The hybridization mixture consisted of 2.5 µ l of each of the differentially labeled DNAs plus 25 µ l of hybridiza- tion buffer provided in the kit. This mixture was dena- tured at 80°C for 10 min. at 80°C, followed by incubation at 37°C for one hr. Five µ l of this probe were Page 2 of 7 (page number not for citation purposes)