Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.

ADAMs and ADAMTSs are multi-domain proteins characterised by the presence of both metalloproteinase and disintegrin-like domains. ADAM proteins are usually type 1 transmembrane proteins, and ADAMTSs are secreted from cells. The dysregulated expression of ADAMs and ADAMTSs has been reported in a wide range of human cancers, where, in many cases, they are implicated as positive regulators of cancer progression. Proteolytically active ADAMs act as ectodomain sheddases, which release extracellular regions of membrane-bound proteins (e.g., adhesion molecules, growth factors, cytokines, chemokines and receptors). Certain ADAMTSs break down extracellular matrix (ECM) proteoglycans (e.g., aggrecan, brevican and versican). Through these actions they are able to sculpt the tumour microenvironment and modulate key processes involved in cancer progression, including cell proliferation, migration and angiogenesis. Members of both groups of protein can also act to inhibit or slow cancer progression: ADAMs can interact with specific integrins to elicit inhibitory effects on cancer dissemination, and certain ADAMTSs possess antiangiogenic activity, which prevents an increase in tumour size. This review covers recent developments in the involvement of ADAM and ADAMTS proteins in human cancer.

Abstract.Little is known about the expression or role of ADAMTS-1, -4 and -5 and their endogenous inhibitor TIMP3 in the liver in physiological and pathological conditions. Their expression was, therefore, investigated in the hepatocellular carcinoma cell lines HepG2 and HuH-7 using qRT-PCR and western blotting, and their cellular localisation by immunocytochemistry. Cytokine treatments were used to assess mRNA and protein modulation. ADAMTS-1, -4, -5 and TIMP3 mRNA and protein were detected in both HepG2 and HuH-7 cells. IL-1β and IL-6 treatments significantly modulated ADAMTS-1 mRNA expression and IL-1β treatment ADAMTS-4 mRNA expression in HepG2 cells. Modulations of mRNA by ≥5-fold did not translate to increased protein expression. This study showed that ADAMTS-1, -4, -5 and TIMP3 were expressed at differential levels in hepatocellular carcinoma cell lines. The pro-inflammatory cytokines IL-1β, TNF-α or IL-6 induced changes in mRNA expression, although these did not translate to the protein level. IntroductionHepatocellular carcinoma (HCC) is the most common primary malignant tumour occurring in the liver which accounts for 80-90% of all primary liver cancers (1). In many cases, the neoplastic transformation of hepatocytes results from accumulation of genetic changes during enhanced cell proliferation in the injured liver in response to paracrine growth factor and cytokine stimulation (2). HCCs have a considerable capacity for vascular invasion, with metastasis and recurrence being the major factors associated with the poor prognosis of HCC (3).Proteolytic modification of cell surface proteins and extracellular matrix (ECM) plays a pivotal role in cancer development and metastasis. Proteolytic enzymes are involved in several processes, at the cellular and organism level, which are dysregulated in cancer, e.g. cell adhesion and migration, cell invasion and angiogenesis. In particular, they mediate tumour invasion at several stages, e.g. detachment of cells from the primary tumour, crossing vessel walls and extravasation into target organs. This involves attachment of oncogenically transformed cells to the ECM followed by its degradation and movement of invading cells through the damaged ECM (4,5).Expression and activity of several classes of proteolytic enzymes have been shown to be modified in cancer, including recently discovered a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). There are 19 ADAMTSs which can be involved in pathological processes such as ECM breakdown and angiogenesis (6). They consist of several domains: the prodomain, metalloproteinase domain, disintegrin-like domain, cysteine-rich domain, thrombospondin type-I repeats (TSRs) and a spacer domain. Several ADAMTSs also contain an additional unique domain(s). They are synthesised as zymogens and after proteolytic processing at the N-terminus to remove the signal sequence and prodomain, they are secreted from cells. For most ADAMTSs this is an important step in their activation. ADAMTSs may also undergo C-terminal proc...

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