## 4 Discussion  
### Unusual Sub-Cellular Locations Reached by Specific Forms of Cathepsins  
Over the last several decades, various reports have suggested an increased proteolytic activity due to over-expression of cysteine cathepsin in distinct types of malignancies such as breast, thyroid, prostate, and colon carcinoma [5; 8; 13; 14; 32; 33; 43]. Additionally, the presence of these proteases was determined to be beyond the canonical endo-lysosomal compartments, considering excessive secretion of cathepsin into the extracellular space and biosynthesis of specific forms and variants that are mis-sorted to reach the nucleus, or even the mitochondrial matrix as well as leakage out of endolysosomes [9; 11; 12; 30; 35; 44; 45; 46; 47; 48]. For instance, cathepsin B secretion at the basal plasma membrane domain and nuclear localization of an N-terminally truncated cathepsin B variant was observed in thyroid carcinoma [9; 21; 49]. Various transcripts of cathepsin B can be derived from the _Ctsb_ gene due to exon skipping, by usage of alternative transcription initiation sites or by alternative splicing of pre-messenger RNAs [12]. Both full-length and exon 2-lacking transcripts can encode the same protein because the canonical start codon is present in exon 3. Alternatively, transcripts lacking exons 2 and 3 can be translated by using an alternative start codon present in exon 4. However, transcripts lacking exons 2 and 3 encode an N-terminally truncated form of cathepsin B. Such transcripts code for a protein that is lacking the signal peptide and the first 34 amino acid of the propeptide, thereby resulting in the biosynthesis of a cathepsin B-specific form translated in the cytoplasm [4; 21; 25]. Absence of the propeptide, and with this its inhibitory and chaperoning capability, may affect the folding and subsequent activation of such a cathepsin form [12; 45; 50; 51]. There are, however, clear indications from our group's studies that N-terminally truncated cathepsin  
Figure 8: HCT116 cell cycle progression remains unaltered upon cathepsin B inhibition and is independent of the expression of cathepsin B-EGFP chimeras. **(A,B)** HCT116 cultures synchronized in G1/G0 by serum-starvation were quantified FACS analyses. Proportional distribution of cells in different cell cycle stages in cultures subjected to cathepsin B activity inhibition by CA074 treatment is depicted **(A, left panel)**, while cultures were investigated at 24 h post-transfection with _ph_CB-EGFP (**B**, left panel). The percentages of cells in the G1/G0, S, and G2/M phases of the cell cycle were also determined at 10 and 16 h release from the G1/G0 arrest (**A,B**, middle and right panels, respectively). Note that the typical distribution of non-treated cultures is 85% in G1/G0, 10% in S, and 5% in G2/M at 10 h, while 37%, 32%, and 31% were in the respective phases at 16 h (see [35]). Expression of full-length cathepsin B or inhibition of cathepsin B activity had no effect on HCT116 cultures progressing through the cell cycle. B forms with a molecular mass of 40 kDa may fold properly and eventually even gain proteolytic activity, at least in thyroid carcinoma cells [9]. In the present study, we show differential localization and elevated levels of cathepsin B in adenocarcinoma cells of TMA when compared to the cells in adjacent normal tissue (Figure 1). Similarly, the CRC cell line HCT116 exhibited an enhanced and, in particular, nuclear localization of cathepsin B in comparison with the normal intestine epithelial cell line IEC6 (Figure 2). HCT116 cells expressed a distinct molecular form of cathepsin B characterized by a molecular mass of 40 kDa (Figure 3) that would be consistent with an N-terminally truncated form of cathepsin B that lacks the signal peptide, hence rendering the nascent chain ER-entry incompetent. As a result, the 40-kDa variant of cathepsin B would be translated in the cytoplasm and could be targeted to the nucleus of CRC cells due to productive folding.