Cervical cancer is one of the major causes of gynecological death worldwide. The degree of cancer progression of is categorized into different stages. Therapy is given depending on the stage of the cancer. Radiation is the main therapy in the advanced stages. Unfortunately, radiation therapy also causes severe damage to normal tissue. A better understanding of the cellular responses to radiation is needed to improve therapy.
Analysis of cell material from patients with cervical cancer can show differences in gene expression. Upregulated expression of the CKS2 gene is associated with high malignancy and poor patient survival. CKS2 could serve as a therapeutic marker and a possible new target for chemotherapeutic drugs. Determining the role of the CKS2 protein in radiation response may therefore contribute to improve therapy.
Two immortalized cervical carcinoma cell lines, Hela and Siha, were used in the experiments in this thesis. Preliminary work included establishing cell line characteristics with respect to growth, plating efficiency and radiosensitivity. Effect of radiation on cell cycle distribution was determined 24 hours after various doses and at different times after irradiation with 8Gy. A method for detecting CKS2 protein content in the cell was developed. The analyses were performed with flow cytometry. A developed staining procedure allowed us to observe the CKS2 protein content in the different phases of the cell cycle individually. Attempts were made to localize the CKS2 protein in the cells. Potential changes in CKS2 protein conformation in the cell cycle were also investigated.
The doubling time was 22 hours for Hela cells and 34 hours for Siha cells. The plating efficiency found for Hela was 49 % and for Siha 45 %. Hela cells were found to be more radiosensitive than the Siha cells, and the difference was significant even at low radiation doses. Further results show that the cervical carcinoma cells from both cell lines were arrested in G2 phase 4 hours after irradiation. The fraction of cell arresting in G2 was increasing with increasing radiation doses. The lowest radiation dose that caused a significant increase in cell fraction in G2-M in both cell lines was 4 Gy. The G2 arrest was abolished 30 and 48 hours after irradiation for Hela and Siha cells, respectively, and the cells reentered the cell cycle.
The CKS2 protein content increased in all phases for both cell lines 6-24 hours after irradiation, but the increase was only significant in M phase. In Siha cells, there was no change in CKS2 protein content at times after 24 hours. For Hela cells there was a decrease followed by a second significant increase after 48 hours. In Siha cells, a significant increase in CKS2 protein content was also detected in G2 phase after 30-48 hours. An increase in CKS2 protein content in M phase was seen in Hela cells with increasing radiation doses. The first significant increase was detected after 4 Gy, and a further increase was detected after 10 Gy. There was also an increase in G2 phase after 4 Gy, from which there was no further change. In Siha cells, a significant increase in CKS2 protein content is only detected in M phase after 8 Gy.
Linking CKS2 protein content to cell cycle distribution was attempted, but further research must be done to clarify the role of CKS2 in radiation response. The localization of the CKS2 protein in the cell was illustrated. Findings that indicated conformational changes between interphase and M phase were also presented. The results can provide useful information for further work with the cervical carcinoma cell lines and their responses to radiation. The CKS2 findings may contribute to a better understanding of the functions of this protein in the cell. As a small part of a larger picture, this work could have an impact on improving therapy for cervical cancer patients.