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Tumors have been known to contain a small population of cancer stem cells that initiate tumor growth and promote tumor spreading. CD133 alone or in combination with other markers is currently being used for identification and isolation of the putative cancer stem cell population from malignant tumors. CD133 is a member of the transmembrane glycoprotein family and was initially described as a surface antigen specific for human hematopoietic stem cells.(1,2) Although its biological function remains largely unknown, CD133 has been recognized as a stem cell marker for cancerous tissues. Indeed, CD133 alone or in combination with other markers is currently used for identification and isolation of the putative cancer stem cell population from malignant tumors, as well as cell lines of brain, (3,4) prostate, (5) liver, (6,7) pancreas, (8) colon (9,10) and melanoma.In a recent report, CD133 was also used to isolate cancer stem cells from lung cancer.(12) The lung cancer CD133 + subpopulation was able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). When injected into immunocompromised mice, they readily generated xenograft tumor phenotypically identical to the tumor derived from the original unsorted cancer cells. Upon differentiation, lung cancer CD133 + cells acquired the specific lineage markers, while losing the tumorigenic potential together with the CD133 expression. (12) However, in one recent study on different in vitro phenotypes of primary glioblastomas, although most primary cells grown as tumor spheres were driven by CD133 + cells, four of the 15 cell lines grown adherently were driven by stem cell-like CD133 -tumor cells (13) and both the CD133 + and CD133 -tumor cells from these cells were similarly tumorigenic in nude mice in vivo.(13) It was also reported that both CD133 + and CD133 -metastatic colon tumor subpopulations formed colonospheres in cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. (14) Furthermore, both CD133 + and CD133 -cells displayed similar frequencies of stem cell-like properties in DAOY medulloblastoma cell line.(15) Given the contradiction regarding the role of CD133 as a true cancer stem cell marker, we investigated whether the CD133 + subpopulations of a non-small cell lung cancer A549 cell line and a small lung cancer H446 cell line characteristically resemble the cancer stem cells more closely than the CD133 -subpopulations. Materials and MethodsCell culture. Human lung cancer cell lines A549 and H446 were cultured in RPMI-1640 (HyClone, Logan, UT, USA) and 10% fetal bovine serum (Tian Jin Hao Yang Biological Manufacture, Tian Jin, China) in a tissue culture incubator at 37°C under 5% CO 2 and 100% humidity. To determine the clonogenicity and regeneration ability of single cells, colony formation assay was carried out as described previously (16) with some modifications. CD133 + and CD133 -cells of A549 and H446 were resuspended in fres...

Feces are increasingly used as sources of DNA for genetic and ecological research. This paper describes a new method for isolation of DNA from animal feces. This method combines multiple purification steps, including pretreatment with ethanol and TE, an inhibitor-absorber made of starch, the CTAB method, the phenol-chloroform extraction method, and the guanidinium thiocyanate-silica method. The new method is efficient according to PCR results of 585 fecal samples from 23 species and costs much less than the commercial kits. The protocol can be tailored to the specific purpose of examining different diets of animals and can be performed with routine laboratory reagents.

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