MDR genes, ABC transporters, a review: superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance. ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs.

Pgp transports various substrates across the cell membrane including:

Drugs such as colchicine, tacrolimus and quinidine

Chemotherapeutic agents such as etoposide, doxorubicin, and vinblastine

Immunosuppressive agents

HIV-type 1 antiretroviral therapy agents like protease inhibitors and nonnucleoside reverse transcriptase inhibitors.

Regulating the distribution and bioavailability of drugs

Increased intestinal expression of P-glycoprotein can reduce the absorption of drugs that are substrates for P-glycoprotein. Thus, there is a reduced bioavailability, and therapeutic plasma concentrations are not attained. On the other hand, supratherapeutic plasma concentrations and drug toxicity may result because of decreased P-glycoprotein expression

Active cellular transport of antineoplastics resulting in multidrug resistance to these drugs

HSCs and SP cells

Cells of the hematopoietic system develop in the bone marrow from a population of self-renewing stem cell and progenitor cells, hematopoietic stem cells (HSCs). These cells make up only 0.01–0.10% of bone marrow, and a recent focus of stem cell biology has been isolating, characterizing, and using these HSCs to replenish normal bone marrow function. HSCs, ESCs, adult stem cells, and cancer stem cells, have been found to express MDR / ABC transporters at a high level, proportionate to stemness, and to efflux in the sp face tail as below on H dye assays.

HSC sp cells - CD34–/low c-kit+ Sca-1+ Lin–

Goodell and coworkers have described a fluorescence-activated cell sorting (FACS) technique to identify the most primitive HSCs present in human, murine, and primate bone marrow based on their specific ability to efflux the supravital

dye Hoechst 33342.1 This population of HSCs, called “side population (SP) cells”, is able to replenish normal bone marrow function in vivo. These cells have traditionally been identified using a flow cytometer fitted with a 350-nm UV laser.

Recently, however, Telford and Frolova determined that SP cells can also be identified by exciting the Hoechst dye with a 375-nm near UV laser.2 Osawa et al, developed an alternative method to identify and purify the HSCs based on cell surface marker expression.3,4 This combination of markers is composed of CD34–/low c-kit+ Sca-1+ Lin–, and the cells are referred to as CD34KSL. Using these markers, it is possible to achieve a high degree of HSC enrichment of an extremely small and homogeneous population of HSCs with a long-term capability for self renewal (LT-HSCs).

The ability to characterize and isolate stem cell populations and monitor the dynamic changes that occur in both intracellular and surface proteins during differentiation is essential to understanding underlying developmental pathways. A main focus in stem cell biology has been isolating, characterizing, and using hematopoietic stem cells to replenish normal bone marrow function. Cells of the hematopoietic system develop in the bone marrow from a population of selfrenewing progenitor cells known as hematopoietic stem cells (HSCs). Flow cytometry and cell sorting are valuable research tools for using cellular markers to isolate and characterize HSCs. HSCs represent approximately 1 in 103–104 bone marrow cells in the adult mouse. Goodell and coworkers have described a method using dual wavelength flow cytometry, which defines a small (0.01–0.10%) population of the most primitive HSCs that selectively efflux the supravital dye Hoechst 33342 (bis-benzimide) in murine, primate, and human bone marrow, as well as in a variety of other tissues.1 The cells of the population of HSCs identified by this method are called side population (SP) cells. Since the enriched SP cells can be used to replenish normal bone marrow function, it is important that SP cells are functional after sorting. This can be assessed using a colony-forming cell (CFC) assay to investigate the growth of lineage-restricted colonies. Traditionally, these cells have been enumerated by exciting Hoechst-labeled SP cells with the 350-nm line of a UV laser and simultaneously measuring blue and far red light emission. Experiments performed in 2003 by Telford and Frolova at the National Institutes of Health (NIH) determined that SP cells could also be analyzed by exciting the Hoechst (HO) dye with a 375-nm near UV laser.

Cancer, ESC, and Adult Stem Cell Biology – Finding the Common Ground:

Numerous observations made our group and others, in the late 90s (Kukekov, AACR, Sci Abstract, San Diego) and in 2002 (Kukekov et al), 2003 (Duntsch et al.), and 2005 (Kukekov and Ignatova et al. and USPTO / WPTO patents 2006 -2—12) (see refs below) led to a huge paradigm shift and a stem cell hypothesis for solid cancers.

For example, a host of embryonic antigens and functional proteins (proteins not expressed in adult tissue stem cells or adult somatic cells, being restricted to germ, embryonic, or fetal cells and tissues) were commonly being reported in the literature as being expressed in various types of adult nongerm somatic cancers (Ignatova et al., 2002). Similarly, antigens and structural proteins whose expression in adult tissues and cells was known to be restricted to a certain germ layer (for example, the expression of glial fibrillary acidic protein in glial cells in the nervous system, derived from and restricted to neuroectoderm, and thus ectoderm) were being reported in the literature as being expressed in cancers derived from a different germ layer (Ignatova et al., 2002). Osteosarcomas (originating in bone, and derived from mesoderm) were found to overexpress the microtubule structural protein B-Tubulin III (normally restricted to expression in differentiated neurons derived from ectoderm) (Gibbs and Kukekov et al., 2005). Indeed, the expression of B-Tubulin III in osteosarcoma is a sensitive and specific cancer marker for aggressive malignant osteosarcoma that is highly metastatic (and associated with a poor prognosis). Further, similarities were noted in the differentiation profile of embryonal carcinomas and the normal development of the inner cell mass of a blastocyst, as well as in morphologic similarities between in vitro clonal ESCs, adult stem cells, and cancer spheroid formation from single cells cultured in semisolid media(Skotheim et al., 2005; Okada K et al., 2003; Sperger JM et al., 2003).

Neural stem cells were first isolated from the adult brain and expanded in vitro under specific stem cell promoting culture conditions that simultaneously suppress the growth of non-stem or more differentiated cells (originally designated as the neurosphere culture system) (Kukekov et al., 1997; 1999). The key components of this system are low cell density, serum and anchorage withdrawal (lack of attachment to substrate), and the presence of the pleiotropic growth factors FGF2, EGF, and insulin.

This approach has been used to isolate CSCs from a variety of cancers (referred as the oncosphere culture system), including adult high-grade glioma, breast cancer, osteosarcoma, and others (Ignatova et al., 2002; Al-Hajj et al., 2003; Gibbs and Kukekov et al., 2005). This culture system was used to study many aspects about CSCs and much was learned. Some of the observations made about CSCs in this system are as follows: CSCs grow clonally (each cluster derived from a single cell) into CSC clusters that have many characteristics of stem cells and malignant features of cancer cells, and have high expression of CSC markers, MDR genes, and tumor

Typically higher expression is associated with an stem cell phenotype including: immaturity, unlimited proliferation capability, lack of differentiation or inablity to differentiate, pluripotency, increased expression of MDR genes (ABC transporters), increased motility, more aggressive invasive phenotype, EMT,

increased expression of tumor suppressor genes, decreased sensitivity to DNA damage, and many other biological features. Typically, as expression declines and becomes low to null expression, stem cell features are lost and cell differentiation occurs. This is a one way event, does not reverse, and thus the phenotype is lost.These events lead to differentiation of the cells, and are associated with non-stem phenotypy including: maturity, decreased expression of MDR genes (ABC transporters), decreased motility, decreased tissue invasion potential, limited or fixed proliferative capacity, no EMT capability, decreased expression of tumor suppressor genes, increased sensitivity to DNA damage, and other features of mature restricted terminally differentiated cells.

Slide 1 A. Phase-contrast images of monoclonal sarcospheres suspended in methylcellulose and grown under stem cell promoting conditions (left panel), immediately after allowing sphere to attached to a substrate coated surface (middle panel), and 3 hours after attachment to a substrate coated surface (right panel). Demonstrates the compact, undifferentiated morphology that is typical of this CSC spheroid cluster while in culture, and the changes that occur over time in CSCs when they are exposed to serum and allowed to attach (flattening, proliferation, and migration from the stem cell cluster). B. A high-grade glioma derived (from LN229) CSC sphere cluster shown 3 hours after attachment to a substrate coated surface. Demonstrates high proliferative activity and imaturity in the center of the cluster, and a change in morphology and maturation (as indicated by the increased expression of Btubulin III) in the periphery of the cluster as the cells begin to differente and migrate from the cell cluster. C. A breast cancer derived (from MDAMB231) CSC sphere cluster shown 6 hours after attachment to a substrate coated surface. Demonstrates proliferative and migratory activity filling the plate with cells, as well as high expression of MDR I in the center of the cluster in CSCs, and a change in morphology and decreased expression of MDR in the periphery and beyond the CSC sphere cluster. D. A high-grade glioma derived (from LN229) CSC sphere cluster shown 6 hours after attachment to a substrate coated surface. Demonstrates proliferative and migratory activity filling the plate with cells, as well as high expression of the tumor suppressor protein BAD in the center of the cluster in CSCs, and a change in morphology and decreased expression of BAD in the periphery and beyond the CSC sphere cluster.

Slide 2 FACS of SP cells: Analysis of mouse bone marrow (panel A) gating strategy used to enrich SP/KSL cells (panels B through H) and analysis of the enriched cell population (panels I through K).

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