Biologic characteristics of the side population of human small cell lung cancer cell line H446.
OBJECTIVE
Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs.


METHODS
Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice.


RESULTS
The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P < 0.01, P < 0.01, and P > 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors.


CONCLUSIONS
The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.
Wang
Bo
B
Department of Radiotherapy, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P. R. China.
Yang
Huan
H
Huang
Yu-Zheng
YZ
Yan
Ru-Hong
RH
Liu
Fen-Ju
FJ
Zhang
Jun-Ning
JN
D000071916:AC133 Antigen; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology* / Q000637:transplantation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D058985:Side-Population Cells / Q000378:metabolism / Q000473:pathology* / Q000637:transplantation; D055752:Small Cell Lung Carcinoma / Q000378:metabolism / Q000473:pathology*
C116614:ABCG2 protein, human; D000071916:AC133 Antigen; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015703:Antigens, CD; D014408:Biomarkers, Tumor; C469949:GNL3 protein, human; D006023:Glycoproteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse; D012333:RNA, Messenger; D019204:GTP-Binding Proteins