It has been shown that melanoma cells do not express argininosuccinate synthetase (ASS) and therefore are unable to synthesize arginine from citrulline. Depleting arginine using pegylated arginine deiminase (ADI-PEG20) results in cell death in melanoma but not normal cells. This concept was translated into clinical trial and responses were seen. However, induction of ASS expression does occur which results in resistance to ADI-PEG20. We have used 4 melanoma cell lines to study factors which may govern ASS expression. Although these 4 melanoma cell lines do not express ASS protein or mRNA as detected by both immunoblot and northernblot analysis, ASS protein can be induced after these cells are grown in the presence of ADI-PEG20, but again repressed after replenishing arginine in the media. The levels of induction are different and one cell line could not be induced. Interestingly, a melanoma cell line with the highest level of induction could also be made resistant to ADI-PEG20. This resistant line possesses high levels of ASS mRNA and protein expression which cannot be repressed with arginine. Our study indicates that ASS expression in melanoma cells is complex and governed by biochemical parameters which are different among melanoma cells.

Background: Cisplatin resistance is complex and involves several different mechanisms. Employing cDNA microarray analysis, we have found that cisplatin resistant cells share the common characteristic of increase in ribosomal proteins and elongation factors. We hypothesize that in order to survive cisplatin treatment, cells have to synthesize DNA repair proteins, antiapoptotic proteins and growth-stimulating proteins. Thus, by blocking the translation of these proteins, one should be able to restore cisplatin sensitivity. We have studied the role of CCI-779, an ester analog of rapamycin which is known to inhibit translation by disabling mTOR, in restoring cisplatin sensitivity in a panel of cisplatin resistant cell lines. We have also determined the role of CCI-779 in P-gp1 and MRP1 mediated resistance.

We have previously shown that the multidrug-resistance/P-glycoprotein gene, mdr3/mdr1a, is activated in mouse hepatocellular carcinomas (HCC). In this study, we show that in a number of HCC-derived cell lines (Hepa1c1c, Hepa1c1c-BprC1, and Hepa1-6) mdr3 is expressed at high levels. To investigate transcriptional regulation of mdr3 in these cells, we have isolated a DNA fragment containing the 5' portion of the mouse mdr3 gene and performed a functional analysis of its promoter. Transient transfection assays using various lengths of the promoter sequence to direct expression of the chloramphenicol acetyltransferase (CAT) reporter gene revealed that the sequence located -94 nucleotides upstream from mouse mdr3 transcription start site functions as a negative element in mouse hepatoma cells. A canonical AP-1 binding sequence TGA-GTCA located at -117 is at least in part responsible for the negative effect from the following observations: (i) Alteration of this AP-1 sequence by site-directed mutagenesis enhanced CAT expression. (ii) Expression of CAT reporter gene was elevated when double-stranded DNA containing the AP-1 sequence, but not mutated sequences, was used as a competitor in cotransfection experiment. (iii) Enhancement of the CAT expression was also seen in cotransfection experiments using recombinant plasmid DNA expressing the c-jun/c-fos proteins, which interact with AP-1 sequences. Interestingly, the proximal region of the hamster pgp1 promoter shares striking sequence similarity with that of the mouse mdr3 gene, including the AP-1 site, but the AP-1 site in the hamster promoter serves as a positive regulator. Although previous studies have demonstrated that positive and negative transcription factors can modulate gene expression through interactions with c-jun/c-fos, this is the first study to show that an AP-1 site functions as a negative cis-element in the regulation of gene expression.

In this report we describe a novel gene delivery system using malaria circumsporozoite (CS) protein as a specific ligand. The CS protein covers the entire surface of sporozoites of malaria parasites. Previous studies have demonstrated that intravenously injected CS protein binds specifically to the basolateral surface of hepatocytes within minutes, indicating the high hepatocyte specificity of CS protein. This characteristic of CS protein prompted us to explore the possibility of using this protein as a liver-specific ligand for hepatic gene delivery vehicle. As an initial step, we investigated the efficacy of CS protein-mediated gene transfer into primary hepatocytes as well as established cell lines. Recombinant CS proteins were chemically conjugated to poly(L-lysine). The CS conjugates were complexed with recombinant plasmid DNA carrying a reporter gene. When the DNA complex was used to transfect primary hepatocytes, a very low level of expression of the reporter gene was observed. The level of expression was greatly enhanced when the cells were cotransfected with adenovirus, which presumably releases the internalized DNA from endosomal entrapment. The CS-mediated gene transfer into the cells required region II+, an evolutionarily conserved amino acid sequence conferring the binding of CS protein to its receptor. CS protein also efficiently mediated gene transfer into a number of cell lines, i.e. HepG2, HeLa, NIH3T3, and K562, but not HL-60, which contains low levels of receptor. Thus, the CS conjugate can be used to deliver DNA into many different cultured cells. Most importantly, the CS conjugate has a potential to be further developed into a liver-specific gene delivery vehicle in vivo.

P-glycoprotein has been shown to be responsible for multidrug resistance in mammalian cells. However, its physiological roles in normal cells are not known. The gene encoding this protein has been shown to express at a relatively high level in human digestive tracts. In the present study, in situ hybridizations were employed to determine the expression of this gene in gastrointestinal tissues. Epithelial cells in the villi of small intestine, colon, and stomach were rich in the P-glycoprotein gene transcript. Observations were consistent with the idea that the P-glycoprotein plays a role in detoxification by pumping potentially harmful compounds into the lumen of digestive tracts in animals.