This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As described in our last report, human lysyl oxidase-like 2 protein (HLOXL2) has been implicated in the metastasis of breast cancer cells and in the deposition of large amounts of collagen fibers. We anticipate therapeutic agents against HLOXL2 will be useful in preventing cancer from spreading and will reduce the recurrence of cancer following surgical removal of breast tumors. In order to develop these drugs, it is important to understand the basic biochemical and physiological functions of HLOXL2 and closely related proteins. To date, few biochemical studies have been reported for HLOXL2. Because it is difficult to work on human proteins (i.e., limited amounts, availability, inability to conduct genetic experiments), we have chosen to work with the fruit fly Drosophila melanogaster proteins. The genome of the fruit fly has two genes (dloxl1 and dloxl2) that encode proteins very similar to HLOXL2.
We aim to characterize these fly proteins and use the results to understand the function of human LOXL2 and its role in cancer metastasis. Initially, we determined that the two genes are differentially expressed in different tissue at distinct developmental stages. dloxl1 is expressed robustly in prepupae while dloxl2 is only expressed in adult flies. Tissue specific analyses have been used to demonstrate that the expression of dloxl1 is confined to salivary glands. Surprisingly, we found that dloxl2 was expressed only in adult heads and not in the bodies. To determine the tissue specific expression of dloxl2 in adult heads, we isolated polyA mRNAs from eya2 (eye absent) mutant flies We could not detect dlox2 expression in the eya2 samples, strongly suggesting that dloxl2 is expressed in the eye. We have begun to produce polyclonal antibodies specific for each of the Drosophila LOXL proteins. We have been able to obtain an antibody against a DlOXL2 peptide in the LOX core domain. The antibody recognizes a ~52 kDa band from bacteria that express a GST-tagged DLOXL2 LOX core domain but not from bacteria expressing a similar GST fusion protein from DLOXL1. We are currently testing the antisera on blots of Drosophila tissues. In addition, we are in the process of generating antibodies specific for DLOXL1 using a GST fusion of the entire DLOXL1 coding sequence, because our attempts to use DLOXL1-specific peptides have thus far have not been successful. Our immediate plan is to focus on defining the subcellular localization of DLOXLs by in situ immunostaining, developing loss-of-function-flies that lack DOXL1 to understand the physiological importance of the gene and the biochemical characterization of the native DLOXLs.
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