Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alone and in combination with ionizing radiation in a panel of established human melanoma cell lines as well as in normal human melanocytes. Betulinic acid strongly and consistently suppressed the growth and colony-forming ability of all human melanoma cell lines investigated. In combination with ionizing radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human melanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the expression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermore, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiotherapy. We conclude that the strictly additive mode of growth inhibition in combination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting different target cell populations.

The bcl-2 proto-oncogene is frequently expressed in human cancer. Although bcl-2 was first cloned as the t(14;18) translocation breakpoint from human follicular B-cell lymphoma, it has become apparent that many cell types express bcl-2 because of transcriptional regulation. As such, several transcription factors have been demonstrated to activate expression of bcl-2, including NF-B. We investigated the role of NF-B1 (p50) homodimers in the expression of Bcl-2 in two murine B-cell lymphoma cell lines: LY-as, an apoptosis-proficient line with low Bcl-2 protein expression and no nuclear NF-B activity, and LY-ar, a nonapoptotic line with constitutive p50 homodimer activity and 30 times more Bcl-2 protein expression than LY-as. We found that nuclear p50 homodimer activity correlated with Bcl-2 expression in these cell types and identified several sites within the bcl-2 5-flanking region that p50 was capable of binding. In vitro transcription revealed that recombinant p50 enhanced the production of run-off transcripts from the bcl-2 P1 promoter. Additional in vitro transcription experiments suggested the sites by which p50 afforded this effect. We conclude that the p50 homodimer is capable of transcriptional activation of the bcl-2 gene and suggest that its nuclear activity contributes to the expression of bcl-2 in LY-ar cells.

Understanding the damage of DNA bases from hydrogen abstraction by free OH radicals is of particular importance to understanding the indirect effect of ionizing radiation. Previous studies address the problem with truncated DNA bases as ab-initio quantum simulation required to study such electronic spin dependent processes are computationally expensive. Here, for the first time, we employ a multiscale and hybrid Quantum-Mechanical-Molecular-Mechanical simulation to study the interaction of OH radicals with guanine-deoxyribose-phosphate DNA molecular unit in the presence of water where all the water molecules and the deoxyribose-phosphate fragment are treated with the simplistic classical Molecular-Mechanical scheme. Our result illustrates that the presence of water strongly alters the hydrogen-abstraction reaction as the hydrogen bonding of OH radicals with water restricts the relative orientation of the OH-radicals with respective to the DNA base (here guanine). This results in an angular anisotropy in the chemical pathway and a lower efficiency in the hydrogen abstraction mechanisms than previously anticipated for identical system in vacuum. The method can easily be extended to single and double stranded DNA without any appreciable computational cost as these molecular units can be treated in the classical subsystem as has been demonstrated here.

Erythropoietin is well known for its role in the control of erythropoiesis, where it acts by binding to its cognate receptor (EpoR) on the surface of erythroid progenitor cells. Here we present the novel finding that the EpoR is also expressed in cells of the melanocytic lineage. It is expressed in transformed cell lines established from normal melanocytes and also in established human melanoma cell lines derived from melanoma metastases, but not in normal primary human melanocytes. The analysis of individual subclones isolated from spontaneously transformed melanocytes revealed that approximately 50% of all the clones examined expressed the EpoR. Further analysis of the individual growth characteristics of EpoR-positive and EpoR-negative clones indicated that, under standard cell culture conditions, expression of the receptor did not affect cell growth. Expression of this receptor is consequently most likely driven by an event that is associated with, but not absolutely required for, the transformed phenotype. While the definite function of this receptor in melanoma cells is still unknown and additional studies are required, our findings support the hypothesis that the EpoR may serve as a progression marker for human melanoma. This observation might be useful in the early diagnosis of melanoma.

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