Analysis of hypoxia-inducible factor-1alpha accumulation and cell cycle in geldanamycin-treated human cervical carcinoma cells by laser scanning cytometry.
BACKGROUND
Tumor hypoxia has been linked to increased disease aggressiveness and poorer treatment outcomes, and the transcription factor hypoxia-inducible factor-1 (HIF-1) has been identified as the key molecule mediating the cellular response to hypoxic microenvironments. The alpha-subunit of this factor is accumulated under hypoxia and rapidly degraded during re-oxygenation, rendering the reliable measurement of HIF-1alpha a difficult task. Heat shock protein 90 (Hsp90) is an essential protein that controls the activity, turnover, and trafficking of a variety of other proteins including HIF-1alpha and cell cycle regulators. Hsp90 inhibitors like geldanamycin therefore have the potential to target tumor-cell survival by at least two mechanisms, compromising the accumulation of HIF-1alpha and cell proliferation.


METHODS
We describe here the simultaneous measurement of HIF-1alpha and cell cycle parameters by laser scanning cytometry (LSC) after exposure of two different human cervical carcinoma cell lines to hypoxia and geldanamycin.


RESULTS
Our analysis demonstrates that the cell lines react to hypoxia and drug treatment in a distinct way, with SiHa being more affected by low oxygen concentrations than is ME180, which was more sensitive to geldanamycin treatment. Both cell lines respond to geldanamycin with a G(2)/M-phase arrest and a decrease in HIF-1alpha accumulation. Cell death due to geldanamycin occurs in association with mitosis, presumably through mitotic catastrophe.


CONCLUSIONS
Our results indicate that LSC can significantly contribute to the evaluation of in vitro drug effects particularly with respect to tumor hypoxia and the measurement of HIF-1alpha.
Schwock
Jörg
J
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Geddie
William R
WR
Hedley
David W
DW
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D016227:Benzoquinones; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D016195:G2 Phase / Q000187:drug effects; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis*; D047029:Lactams, Macrocyclic; D047569:Laser Scanning Cytometry; D008938:Mitosis / Q000187:drug effects; D011809:Quinones / Q000494:pharmacology*; D002583:Uterine Cervical Neoplasms / Q000737:chemistry* / Q000473:pathology* / Q000503:physiopathology
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; C497442:HIF1A protein, human; D018841:HSP90 Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D011809:Quinones; C001277:geldanamycin