To predict which of those patients are at a higher risk for progression to cervical cancer, it is necessary to look for new simple and low cost comple mentary prognostic methods. In the current study we used a very simple method based on the capacity of human serum to induce apoptosis in Jurkat cells in an effort to identify a prognostic marker or method which could assist in determining or predicting full article the risk of developing cervi cal cancer. Methods Patients The study group consisted of 22 women with clinical and histopathological diagnosis of squamous cell carcinoma of the cervix, 21 women with CIN 1 and 20 healthy female volunteers. The age of cancer patients, CIN 1, and control group ranged from 30 to 83, 22 to 55, and 22 to 46 years, respectively.
It is important to mention that patients included in this study did not receive any prior Inhibitors,Modulators,Libraries treatment. All patients signed an informed consent form approved by the Ethical Committee of the Instituto Mexicano del Seg uro Social. Serum samples Sera from untreated patients with cervical intraepithelial Inhibitors,Modulators,Libraries neoplasias grade 1 and with cervical cancer were obtained at Centro M��dico Nacional de Occidente IMSS. Control serum samples were obtained from healthy donors. Inhibitors,Modulators,Libraries All serum samples were obtained from peripheral blood by venipuncture after centrifugation at 2000 rpm for 15 min utes, aliquoted and stored at 70 C until used. Cell Culture JURKAT and JURKATR were cultured routinely in RPMI 1640 medium, supplemented Inhibitors,Modulators,Libraries with 10% fetal calf serum, 100 U Inhibitors,Modulators,Libraries mL penicillin and 100 g mL streptomycin. All products mentioned before were obtained from GIBCO Invitrogen Corporation.
Cultures were maintained at 37 C in a humidified atmosphere with 5% CO2. JURKATR is a JURKAT variant resistant to CD95 mediated apoptosis obtained after continuous exposure to agonistic Apo 1 antibody. Exposure of sera more to Jurkat cells and apoptosis detection To test the rate of apoptosis induced by sera from the dif ferent groups, JURKAT or JURKATR cells were seeded at a density of 2. 5 105 cells per well in 1 mL RPMI medium in 6 well plates. Afterwards, 0. 5 mL of serum from the dif ferent women was added. After 3 days of incubation, cell death was measured by flow cytometry using propidium iodide and Annexin V Fluos as recommended by the manufacturers. For each sample, 10,000 events were analyzed in an Epics XL MCL Flow Cytometer using the FL 1 and FL 3 detector filters. Each serum was tested 3 to 5 times in independent experiments. Induction of apoptosis by CD95L To corroborate the sensitivity of JURKAT and JURKATR cells to CD95L induced apoptosis, 3. 5 105 cells were seeded in 6 well plates and exposed to 5 g mL anti Fas human, activating clone CH11, in a final volume of 1 mL RPMI medium.