Bruton's tyrosine kinase (Btk), 3 a member of the Tec family of protein-tyrosine kinases, has been shown to play important roles in B cell development, activation, and survival. Mutations in Btk are known to lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice (1). These diseases are characterized by a block in B-lymphopoiesis and CD5ϩ B-1 cell generation, defects in B cell receptor (BCR) signaling, and impairment in humoral immune responses to certain types of T cell-independent antigens (2). Structurally, Btk contains multiple protein-protein interaction domains. It has a pleckstrin homology domain for membrane localization following its activation, as well as Src homology 2, Src homology 3, and proline-rich domains for binding other signaling molecules. In addition, it possesses multiple tyrosine phosphorylation sites. Hence, Btk is postulated to play a key role in signal transduction processes.

Hexamethylene Bisacetamide (HMBA) is a hybrid polar compound originally developed as a differentiation inducing agent. We show in this study that HMBA can inhibit activation of several NFκB target genes in both lung and breast cancer cell lines. Furthermore, consistent with its ability to inhibit NFκB function, HMBA can also sensitize cells to apoptosis. We show that HMBA mediates inhibition of the Akt and ERK/MAPK cascade, both of which are critical for cell survival and proliferation and are well known regulators of NFκB activation. We also show that PTEN negative breast cancer cells which have hyper activation of the PI3K/Akt pathway show increased sensitivity to growth inhibitory effects of combination of HMBA and TNFα. Furthermore, HMBA can decrease the kinase activity of the IKK complex leading to defective phosphorylation of IκBα and Ser536 of p65. This study gives mechanistic insight into the mechanism of action of HMBA, provides the rationale for the potential use of HMBA in combination with various existing kinase inhibitors in combination therapy and also suggests useful biomarkers for monitoring tumor response to HMBA.

Overexpression of mutant p53 is a common finding in most cancers but testicular tumours accumulate wild-type p53 (wtp53). In contrast to the accepted concept that p53 homozygous mutant mice do not accumulate mutant p53 in normal cells, our study on a mutant p53 mouse model of Li-Fraumeni syndrome harbouring the hot-spot p53R172H mutation described an elevated level of mutant p53 in non-cancerous mouse tissues. Here we use detailed immunohistochemical analysis to document the expression of p53R172H in mouse testis. In developing and adult testes, p53R172H was expressed in gonocytes, type A, Int, B spermatogonia as well as in pre-Sertoli cells and Leydig cells but was undetectable in spermatocytes and spermatids. A similar staining pattern was demonstrated for wtp53. However, the intensity of wtp53 staining was generally weaker than that of p53R172H, which indicates that the expression of p53R172H can be a surrogate marker of p53 gene transcription. Comparing the responses of wtp53 and p53R172H to irradiation, we found persistent DNA double-strand breaks in p53R172H testes and the formation of giant spermatogonia (GSG) following persistent DNA damage in p53R172H and p53-null mice. Strikingly, we found that p53R172H promotes spontaneous formation of GSG in non-stressed p53R172H ageing mice. Two types of GSG: Viable and Degenerative GSG were defined. We elucidate the factors involved in the formation of GSG: the loss of p53 function is a requirement for the formation of GSG whereas DNA damage acts as a promoting trigger. The formation of GSG does not translate to higher efficacy of testicular tumorigenesis arising from mutant p53 cells, which might be due to the presence of delayed-onset of p53-independent apoptosis.