Chromosome 8 aberration and c-myc amplification have been suggested as playing important roles in the development of different human cancers. Using fluorescence in situ hybridization (FISH), chromosome 8 polysomy and c-myc amplification can be detected in cells from bladder cancer. We investigated the correlation of chromosome 8 polysomy, c-myc gene alteration and p53 deletion with histopathological parameters. Twenty-four tumors obtained from patients with bladder cancer were analyzed by interphase cytogenetics using FISH with chromosome 8 and 17 centromere probes together with an YAC clone covering the c-myc locus and three cosmid DNA probes covering the p53 locus. Chromosome 8 polysomy was found in 12 tumors. The average copy number of chromosome 8 centromere signals were significantly higher in high grade and stage, cancers. Also the c-myc copy gain and p53 deletion were significantly correlated with grade as well as stage (p<0.05, in both cases). Both polysomy 8 and c-myc copy gain were significantly correlated with p53 deletions (p<0.01) and DNA ploidy (p<0.001). On the contrary there was no significant correlation between c-myc protein over-expression and c-myc gene amplification. These results may indicate that alteration of chromosomal regions on 8q and 17p, including c-myc and p53 genes, may be linked to progression of bladder cancer.
In order to investigate the significance of p53 deletion, 42 specimens of transitional cell carcinoma were analyzed by interphase cytogenetics with a fluorescence in situ hybridization technique and compared with clinicopathological and cytochemical parameters. In total, 27 (64%) and 16 (38%) specimens demonstrated p53 deletion and overexpression, respectively. The p53 deletion was significantly correlated with grade (P < 0.01), stage (P < 0.05), S-phase fraction (P < 0.05), and DNA ploidy (P < 0.01), while p53 overexpression correlated only with grade (P < 0.05). The close correlation of p53 deletion with clinicopathological parameters suggests p53 deletion to be of clinical importance to indicate the malignant potential of human urothelial tumors.
The application of cytokines for immunotherapy is frequently hampered by undesirable side effects. To avoid systemic effects, cytokines can be directly expressed in the target cells by using gene transfer. However, the uncontrolled cellular secretion of cytokines could still exert some undesirable bystander effects. Therefore, it is important to develop additional methods for a more restricted administration of cytokines. Recently, using the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), we have demonstrated that cytokines can be targeted to different subcellular compartments as stable and biologically active proteins. This model could be used as a method of highly restricted administration of cytokines. Here, as model for the proof of principle, we have used a cell line (DA-3) strictly dependent on mGM-CSF for growth and demonstrated that these cells acquired autonomous growth after gene modification with plasmids encoding either extracellular or intracellular forms of mGM-CSF. Cell lines expressing secreted forms of mGM-CSF displayed the highest rates of autonomous growth and released substantial amounts of mGM-CSF. However, cell lines expressing intracellular forms of mGM-CSF also acquired autonomous growth induced by a mechanism of restricted autocrine stimulation and did not release detectable mGM-CSF to the extracellular medium. Cocultivation experiments of DA-3 cell lines expressing intracellular mGM-CSF with unmodified cells showed that there was no activation of the bystander cells. Taken together, these results support the concept that genes encoding intracellular cytokines may be used to provide the desired effect of cytokines on the target cells while avoiding the side effects of their uncontrolled secretion.
Comparative genomic hybridization (CGH) was applied to screen the genetic events in six invasive urinary bladder cancers. These cases were also studied by flow cytometry (FCM) and fluorescence in situ hybridization (FISH). Four samples showed partial gain on chromosome 8, with the common region involved was on 8q23-qter. Full or partial deletion on chromosome 2 and 17p in addition to gain on 20q was found in two cases. Interestingly one diploid tumor with low mitotic index, stage and grade showed more genetic aberrations (8 gains and 7 losses) by CGH than other aneuploid tumors with high mitotic index, stage and grade. The numerical chromosomal aberration detected by FISH for chromosomes 7, 8, 9, 10, 11 and 17 were 50% in T1 cases and 100% in T2-T4 cases. FISH was performed on chromosome 8q and 17p to compare and validate the sensitivity of CGH. The agreement was 100% for 8q24 locus and 50% for p53 locus. This indicates that different molecular genetic techniques showed relatively different aspect of genomic aberrations.
It is known that estramustine (EM) accumulates in cells at the G2/M-phase and causes metaphase arrest of various cell types. The inhibitory effect is mediated by interaction with microtubule-associated proteins (MAPs) and/or tubulin. Estramustine-binding protein (EMBP) is a secretory protein which has been found in a number of different tumor cells and has been shown to faciliate the uptake of EM into cells. In this study the efficacy of EM in arresting cells at metaphase was studied, using four different human cell lines; the prostatic cancer cell line DU 145, the breast cancer cell line MDA 231, the colon cancer cell line Colon 320, and the urinary bladder cancer cell line RT4. The cells were incubated with EM at a concentration of 10 micrograms/ml for 24 hours. The data reveal an increase in metaphase arrests in the DU 145 and in Colon 320 cell lines. Both of these cell lines were found to contain high amounts of EMBP using a dot-blot assay. The other two cell lines, MDA 231 and RT4 had undetectable intracellular amounts of the protein and exhibited a low increase in metaphase arrests. The cell lines were analysed regarding S-phase fraction with flow-cytometry (FCM) to exclude the growth rate of the cells as a limiting factor. The results from the FCM confirmed the cytogenic analysis, that is a higher percentage of cells were in the G2/M phase in both the DU 145 and Colon 320 cell line compared to MDA 231 and RT4. EM causes mitotic arrest in those cell lines that contain detectable amounts of EMBP.
Background: Rapamycin was the first inhibitor of mTOR (mechanistic previously named mammalian target of rapamycin, also known as FRAP or RAFT 1) to be discovered and studied as a therapy for a wide variety of diseases.Since the phosphoinositide 3 kinase (PI3K)/Akt (protein kinase B) /mTOR (PAM) pathway is frequently activated/dysregulated in breast cancer, rapamycin was explored as an effective target therapy in breast cancer, however the mode of its action remains unclear, therefore our study aimed at understanding the antitumor effect of rapamycin, using MCF-7 breast cancer cells.Methods: Trypan blue dye exclusion method and MTT assay were performed to determine the IC50 value of rapamycin.Trypan blue dye exclusion method was also used to determine cell growth in untreated versus rapamycin treated MCF-7 cells, these cells were examined for morphological changes using inverted phase microscope.Results: Rapamycin showed inhibitory activity on MCF-7 cell line via cell growth inhibition as determined by the IC50 value.The IC50 value was determined as 75μg/ml measured by trypan blue dye exclusion method and confirmed by MTT assay.Treatment of MCF-7 cells with rapamycim at IC50 resulted in induction of apoptosis up to 41% as determined by both Annexin-V/Propidium iodide dual staining assay and flowcytometry.Direct examination by inverted phase microscope showed that MCF-7 cells treated with rapamycin at IC50 for 72 hrs.resulted in characteristic morphological changes in the form of cell shrinkage and loss of cell contacts, also the cells became rounded and some of them were floating in the medium.Conclusions: This study demonstrated that rapamycin acts as an anticancer drug via inhibition of cell growth in a dose dependent manner as well as induction of apoptosis .However further studies are needed to characterize the mode of action of rapamycin as an anticancer agent.
