Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells.

Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs.

Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line.

BACKGROUND: Bcl-2 and Bcl-XL are anti-apoptotic paralogues that inhibit apoptosis elicited by a wide variety of stimuli, and play critical roles in cancer development and resistance to treatment. Many clinical studies have indicated that expression of these anti-apoptotic proteins in tumours is associated with poor prognosis. It has therefore been assumed that in cells the essential difference between Bcl-2 and Bcl-XL involves regulation of expression and that they are otherwise functionally similar. To examine this issue, we have compared the function of the proteins and of mutants of Bcl-2 and Bcl-XL specifically targeted to different subcellular sites. METHODS: We generated clones of the human breast cancer line MCF-7 stably expressing known amounts of Bcl-2, or Bcl-XL as determined by quantitative immunoblotting. Clones expressing equivalent amounts of wild-type and mutants of Bcl-2 and Bcl-XL with subcellular localization restricted to the cytoplasm, endoplasmic reticulum or outer mitochondrial membrane were studied in both MCF-7 and Rat-1 fibroblasts. In MCF-7 cells we measured the functional activities of these proteins in preventing apoptosis induced by four different agents (doxorubicin, ceramide, thapsigargin, TNF-alpha). Etoposide and low serum were used to compare the effect of Bcl-2, Bcl-XL and mutants located at the endoplasmic reticulum on induction of apoptosis in fibroblasts. RESULTS: We noted both qualitative and quantitative differences in the functional activity of these two anti-apoptotic proteins in cells: Bcl-2 localized to the endoplasmic reticulum inhibits apoptosis induced by ceramide and thapsigargin but not by doxorubicin or TNFalpha, while Bcl-XL at the endoplasmic reticulum is active against all four drugs. In fibroblasts Bcl-2 localized to the ER did not prevent cell death due to etoposide whereas Bcl-XL in the same location did. Finally in MCF-7 cells, Bcl-XL is approximately ten times more active than Bcl-2 in repressing apoptosis induced by doxorubicin. This difference can be manifest as a large difference in clonal survival. CONCLUSION: When examined in the same cellular context, Bcl-2 and Bcl-XL differ substantially in the potency with which they inhibit apoptosis, mediated in part by differences in the inhibition of specific subcellular pathways.

Photodamage to multiple Bcl-xL isoforms by photodynamic therapy with the phthalocyanine photosensitizer Pc 4.

The antiapoptotic oncoprotein Bcl-2 is now a recognized phototarget of photodynamic therapy (PDT) with the phthalocyanine Pc 4 and with other mitochondrion-targeting photosensitizers. Photodamage, observed on Western blots as the loss of the native 26-kDa Bcl-2 protein, is PDT dose dependent and occurs in multiple cell lines, in the cold, and immediately upon photoirradiation. In our initial study, no photochemical damage was observed to Bcl-xL, in spite of its similarity in size, sequence, location and function to Bcl-2. The original study used a commercial anti-Bcl-xS/L antibody. We have revisited this issue by examining Western blots developed using one of three epitope-specific anti-Bcl-xL antibodies from commercial sources, a polyclonal antibody generated to the entire protein, as well as the antibody used previously. All five Bcl-xL antibodies recognized bacterially expressed Bcl-xL, but not Bcl-2, whereas an anti-Bcl-2 antibody recognized Bcl-2 and not Bcl-xL. All five Bcl-xL antibodies recognized at least one protein migrating at approximately 30 kDa; two of the antibodies recognized an additional band, migrating at approximately 33 or approximately 24 kDa. We now observe Pc 4-PDT-induced photodamage to all Bcl-xL-related proteins, except the 33-kDa species, in several human cancer cell lines. The results indicate that, in addition to the expected quantitative differences that may reflect exposure of individual epitopes, the antibodies also detect proteins of different apparent molecular weights that may be distinct isoforms or post-translationally modified forms of Bcl-xL. No evidence for PDT-induced phosphorylation or degradation was observed. Bcl-xL localized to mitochondria was considerably more sensitive to photodamage than was Bcl-xL in the cytosol, indicating that as previously found for Bcl-2, Bcl-xL must be membrane localized to be photosensitive.