Heterogeneity of aldehyde dehydrogenase expression in lung cancer cell lines is revealed by Aldefluor flow cytometry-based assay.
BACKGROUND
We have been interested in studying the roles of two aldehyde dehydrogenases in the biology of lung cancer. In this study, we seek to apply Aldefluor flow cytometry-based assay for the measurement of aldehyde dehydrogenase (ALDH) activity in lung cancer cell lines, which may become a new tool that will facilitate our continued research in this field.


METHODS
Several established lung cancer cell lines were used, including A549 cell line expressing siRNA against aldehyde dehydrogenase class-1A1 (ALDH1A1). Western blot analysis, spectrophotometry assay, and Aldefluor staining were used to measure protein or enzyme activity in these cell lines. For the purpose of measurement of ALDH activity by Aldefluor in cells with known high ALDH levels, cells were mixed 1:10 with immortalized lung epithelial cell line (Beas-2B), which is known to lack ALDH activity. To delineate dead cells, double staining using Aldefluor and propidium iodide (PI) was done. Double staining was also used to detect changes in ALDH activity in two different cell lines after treatment with 4-hydroperoxycyclophosphamide (4-HC).


RESULTS
Our results show a very good correlation between Aldefluor, Western blot, and spectrophotometry assays. Mixing experiments with Beas-2B cells allowed accurate assessment of ALDH activity in A549 cells at baseline and after siRNA expression, thus establishing an approach that facilitates the measurement of very high ALDH using the Aldefluor assay. Aldefluor staining was able to detect heterogeneity in ALDH expression among as well as within the same cell lines and better assess viability after 4-HC treatment when combined with PI.


CONCLUSIONS
Aldefluor assay can be adapted successfully to measure ALDH activity in lung cancer cells and may have the advantage of providing real time changes in ALDH activity in viable cells treated with siRNA or chemotherapy.
Moreb
Jan S
JS
Department of Medicine, Division of Hematology/Oncology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA. morebjs@medicine.ufl.edu
Zucali
James R
JR
Ostmark
Blanca
B
Benson
Neal A
NA
D000444:Aldehyde Dehydrogenase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D000080924:Aldehyde Dehydrogenase 1 Family; D000970:Antineoplastic Agents / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D002277:Carcinoma / Q000188:drug therapy / Q000201:enzymology* / Q000503:physiopathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods* / Q000639:trends; D005456:Fluorescent Dyes; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000503:physiopathology; D011419:Propidium; D034741:RNA, Small Interfering; D050697:Retinal Dehydrogenase; D013194:Staining and Labeling / Q000379:methods
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D011419:Propidium; D003520:Cyclophosphamide; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase; C011272:perfosfamide