Katherine Kravik

Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols

BackgroundEstablished colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth- and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance.ResultsHCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment.ConclusionAll HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols.

Subcellular Localization of the Spindle Proteins Aurora A, Mad2, and BUBR1 Assessed by Immunohistochemistry:

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.

EARLY CELL KINETIC EFECTS OF A SINGLE DOSE OF NARROW‐BANDED ULTRAVIOLET B IRRADIATION ON THE RAT CORNEAL EPITHELUM

Abstract— The right eyes of 40 rats were exposed to a signal erythemogenic dose fo ultraviolet B irradiation (UVB) at 297nm. The irradiation was directed perpenddicualr to the center of the cornea. The left eyes served as controls. The animals were randomly assigned into 10 groups. The labelling index (LI) after pluse labeling the tritiated thymidine and the mitotic rate (MR) after Colcemid administration were registered in the corneal epithelium at predetermined intervals up to 96 h after the irradiation. A mathematical method was used to corealted corresponding corneal areas from the different animals. In the central the LI was considerably reduced up to 36h after the irradiation. The LI increased toward the peripheral cornea and reached normal values at the limbal area. The MR was also reduced up to 36h. However, this reduction was over the entire epithelium. The block in cell proliferation was followed by increased proliferation.

Cell kinetics of conjunctival and corneal epithelium during regeneration of different-sized corneal epithelial defects.

Abstract. Rats with small (diam. 1.7 mm), medium sized (diam. 35 mm) or large (diam. 5.5 mm) corneal epithelial erosions in one eye were killed 1,2 or 4 days after the injury. The proliferative response was evaluated by measuring the labelling index and the mitotic rate in the corneal epithelium and in the adjacent conjunctiva. The small erosions triggered a proliferative response in the cornea only with the maximum response occurring midperipherally. The medium sized erosions induced a higher and more extensive response in the cornea and also a slight increase of the labelling index in the limbal area. The large erosions induced an even more pronounced response in the peripheral cornea and an increase both of the labelling index and the mitotic rate well beyond the limbal part of the conjunctiva. It is concluded that the magnitude and the extent both of the conjunctival and the corneal regenerative response to a corneal abrasion is correlated to the size of the corneal defect. Temporary reduction in the conjunctival epithelial cell number shows that both cells in the limbal and the extralimbal conjunctiva migrate centri‐petally during healing of large corneal wounds. It is suggested that the stem cell theory should be modified. The limbal area is probably an area in which conjunctival epithelial cells or conjunctiva‐derivated cells transform or differentiate to corneal epithelial cells.

The early cell kinetic response during healing of corneal epithelial wounds.

The labeling index and the mitotic rate were measured at 4-hour intervals during the first 24 hours after a central abrasion had been made in the corneal epithelium of six groups of four rats each. The proliferative response was noted in the conjunctival, the limbal, and the corneal epithelium. After 24 hours, the density of epithelial cells was equal throughout the corneal epithelium, but there was only half the normal number of cells. Physiological mechanisms seem strongly to regulate the total number of cells per square unit throughout the healing corneal epithelium, but the nature of these mechanisms is unknown.