S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/ Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21 WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. INTRODUCTIONProteins may exist in several complexes in a spatial and temporal manner to accomplish distinct functions. Analyses of the interacting partners will provide a strong insight into the physiological role of a particular factor. Therefore, it is essential to identify ideally all interacting partners of proteins in vivo to precisely be able to define their biological function. The investigation of protein complexes of solely endogenously expressed proteins avoid the tendency to detect false positive protein-protein interactions of examinations performed in vitro. A multitude of proteins are involved in both the detection and the repair of DNA damages. It is conceivable that some protein complexes involved in these processes are not yet discovered. A severe form of DNA damage that threaten the integrity of the genome are DNA double-strand breaks (DSBs). DSBs can lead to cell cycle arrest or illegitimate DNA rearrangements that can contribute to cell dysfunction, cell death, or carcinogenesis (Hoeijmakers, 2001). Homologous recombination is a major DNA repair pathway by which DSBs are repaired (Lettier et al., 2006).For the identification of specific interacting proteins of S100A11 (S100C, calgizzarin) we used in the present study a proteomic approach comprising mass spectrometry and immunological techniques. S100A11 belongs to the group of S100 proteins that are considered as multitasking proteins involved in several biological processes such as the Ca 2ϩ signaling network, cell growth and motility, cell cycle progression, transcription, and cell differentiation (Schafer and Heizmann, 1996;Donato, 2001;Eckert et al., 2004). It has been proposed that the S100 proteins are involved in...
Annexin A5 is a Ca(2+)-binding protein which is involved in membrane organization and dynamics. As recent data suggest a role of annexin A5 in cancer we aimed to gain more insight into the biological function of endogenous annexin A5 and assessed its possible influence on proliferation and invasion capacity. We downregulated annexin A5 by RNA interference in HaCaT keratinocytes, squamous carcinoma cell line A431 as well as in a primary cell culture of a human oral carcinoma. Hereby, we detected reduced migration and invasion capacity of HaCaT cells which was even stronger in the oral carcinoma. To determine target genes of annexin A5 we used a metastasis specific microarray. Thereby, genes implicated in cell motility including S100A4, TIMP-3 and RHOC were observed to be regulated. These deregulations were confirmed by RT-PCR or western blots, respectively. These observations suggest that the invasion capacity, a main characteristic of tumors, is at least partially regulated by annexin A5 in oral carcinoma.
In oral mucosa lesions it is frequently difficult to differentiate between precursor lesions and already manifest oral squamous cell carcinoma. Therefore, multiple scalpel biopsies are necessary to detect tumor cells already in early stages and to guarantee an accurate follow-up. We analyzed oral brush biopsies (n = 49) of normal mucosa, inflammatory and hyperproliferative lesions, and oral squamous cell carcinoma with ProteinChip Arrays (SELDI) as a non-invasive method to characterize putative tumor cells. Three proteins were found that differentiated between these three stages. These three proteins are able to distinguish between normal cells and tumor cells with a sensitivity of 100% and specificity of 91% and can distinguish inflammatory/hyperproliferative lesions from tumor cells with a sensitivity of up to 91% and specificity of up to 90%. Two of these proteins have been identified by immunodepletion as S100A8 and S100A9 and this identification was confirmed by immunocytochemistry. For the first time, brush biopsies have been successfully used for proteomic biomarker discovery. The identified protein markers are highly specific for the distinction of the three analyzed stages and therewith reflect the progression from normal to premalignant non-dysplastic and finally to tumor tissue. This knowledge could be used as a first diagnostic step in the monitoring of mucosal lesions.
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