Cancer stem cells (CSCs) are a small reservoir of self-sustaining cells with the exclusive ability for self-renewal and tumor maintenance 
. Only a small subset of cancer cells within a tumor have the characteristics of stem cells, and can thus initiate a tumor when transplanted 
. Putative CSCs in acute myelogenous leukemia (AML) were first isolated in 1994 
. With the advances in fluorescence activated cell sorting (FACS), and newly identified cell surface markers such as Sca-1, and CD133, CSCs can be isolated not only from blood cancers, but also from solid tumors 
CSCs in human breast carcinoma have been identified as a rare population of CD44+
. 100 of these cells were able to form new tumors, whereas other tumor cells failed to form tumors in SCID mice. Breast CSCs were also indentified as CD44+
by Ponti et al
. in 2005 
. CSCs in colon cancer were found to be a less than 2.5% of the population with positive CD133 expression 
, and recently defined as CD44+
. Bronchioalveolar stem cells (BASCs) were recently identified as the putative stem cell population for lung adenocarcinoma, and could be isolated as CD45−
cells by FACS 
. CSCs in small cell and non-small cell lung cancer were also reported as a rare population of undifferentiated CD133+
. These were able to self-renew as tumor spheres in serum-free medium, and generate tumor xenografts phenotypically indistinguishable from the original tumor, when injected in SCID mice.
An alternative approach for isolating stem cell populations is through enrichment of side population (SP) cells 
. SP cells are identified by the capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp-1) 
. Since Goodell et al
. first reported that the SP is enriched in hematopoietic stem cells (HSCs)
, SP cells have been identified in a variety of normal and malignant tissues including the lung 
. Bcrp-1 deficient mice lacked the SP in bone marrow yet had no substantial hematopoietic abnormalities 
. Thus, the precise functional relationship between the SP phenotype and stem cell function remains unclear. Both SP and NSP fractions from the DAOY medulloblastoma cell line were capable of reconstituting the original parental population, and only slight clonogenic enrichment was observed in the SP fraction 
Hematopoietic stem cells (HSCs) are reported to reside in the SP fraction of adult mouse bone marrow (BM), however, a recent report showed the coexistence of NSP HSCs that do not significantly differ from SP HSCs in numbers, and self-renewal capacity 
. In addition, the CD34−/low
cell population, which is highly enriched in mouse HSCs, was almost equally divided into the SP and NSP cells. These were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or NSP 
. Further characterization of the SP in cancer cells is therefore required to fully assess the role these cells play in lung cancer.
Recent findings showed that chemotherapeutic drugs, such as doxorubicin and cisplatin, could induce the propagation of CSCs in vitro 
. These cells maintained their self-renewal capacity as demonstrated by growth of tumor spheres in vitro
, and had high tumorigenic and metastatic potential following inoculation into SCID mice. Chemotherapeutic drugs, such as cisplatin and ifosfamide, are also known to depress the expression of proteasomal subunits 
, and down-regulate ubiquitin-proteasome-dependent proteolysis 
. Reduced 26S proteasome activity was found to be a unique feature of CICs (cancer initiating cells) in glioma and breast cancer, that could be used to track CSCs in vivo 
. However, Benzyloxicarbonyl-Leu-Leu-Nle-CHO (LLNle), a γ-secretase inhibitor, was reported to effectively kill human glioblastoma tumor–initiating cells (GBM TIC) in vitro
by inhibition of the 26S proteasome. Therefore, further elucidation of the 26S proteasome activity is required in order to fully determine its role in CSCs and anticancer therapy. Ultimately, this may aid in the identification of novel targets for future therapeutic intervention.
In the present study, we investigated whether lung CSCs solely reside in the side population and whether lung tumor spheres with self-renewal capacity from NSCLC cell lines could be a source for enrichment of lung CSCs.