Cell invasion requires cooperation between adhesion receptors and matrix metalloproteinases (MMPs). Remodeling of the extracellular matrix (ECM)1 is critical for cancer cell invasion and tumorigenesis (1-5). Membrane type matrix metalloproteinases (MT-MMPs) localized to the invasive front of highly motile cancer cells (6, 7) were shown to be directly involved in matrix breakdown (8 -13). A cooperation involving MT-MMPs and cell adhesion receptors is likely to be essential to migrating cells (3, 14 -16). So far, six members of the MT-MMP subfamily have been identified and partially characterized (2, 17-22). MT1-, MT2-, and MT3-MMP strongly contribute to tumor cell invasion (12). Recent studies demonstrated a functional significance and a direct role of MT1-, MT2-, and MT3-MMP in cell locomotion on laminin-5 (11) and three-dimensional collagen type I lattice (8, 9, 12). In addition, MT-MMPs contribute indirectly to cell invasion by activating soluble secretory MMP-2 (23) and MMP-13 (24), which further cleave multiple matrix substrates (2, 5, 25-29).Integrin adhesion receptors dynamically regulate cell-matrix interactions by the binding to matrix proteins and inside-out signaling (30,31). This allows cells to discriminate any subtle alteration of the environment and to adjust cell locomotion accordingly. Direct interactions with multiple transmembrane and cell surface proteins (32) including integrin-associated protein-50 (33), TM4SF proteins (tetraspanins) (34) and tTG (35) further attenuate adhesive and signaling efficiency of integrins.Cell surface tTG (protein-glutamine ␥-glutamyltransferase, EC 2.3.2.13) promotes integrin-dependent adhesion and spreading of cells. By both direct associations with multiple ␤ 1 and ␤ 3 integrins and the binding with Fn, tTG independently mediates the interactions of integrins with Fn (35). The high affinity binding of tTG with Fn specifically involves the 42 kDa gelatin-binding domain of the Fn molecule, which consists of modules I 6 II 1,2 I 7-9 (36). The enhancement of integrin-mediated adhesion and spreading of cells on Fn is independent from the enzymatic activity of surface tTG (35). Intriguingly, reduced expression of tTG has been linked to aggressiveness and high metastatic potential of tumors, whereas overexpression of tTG in fibrosarcomas inhibited primary tumor growth (37, 38). Proteolysis of tTG at the normal tissue/tumor boundary was observed in invasive tumors (38).Here, we report that depending on the structure of the ECM, MT-MMPs are capable of both positively and negatively regulating locomotion of cancer cells. Matrix-dependent proteolysis of surface tTG by MT1-MMP occurs on tumor cells of a diverse tissue origin, thereby representing a general phenomenon and a novel MT-MMP function. Our data suggest an existence of an unexpected link between tumor cell locomotion, the ECM and membrane-anchored MMPs. Regulatory proteolysis of cell surface adhesion proteins by the adjacent MT-MMP molecules is likely to play a significant functional role in cancer cell invasion.

The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the 51 Cr-release assay and IFN-γ ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the 51 Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

Solar ultraviolet radiation is a ubiquitous environmental carcinogen responsible for 500,000 or more new cases of skin cancer in the United States each year (1, 2). Exposure of human cells to natural sunlight leads to the formation of cyclobutane pyrimidine dimers (CPDs), 1 pyrimidine(6 -4)pyrimidone dimers, and their Dewar valence isomers (3). UV-induced DNA photoproducts are currently accepted as important underlying factors in skin carcinogenesis (2, 4). Studies with UVC (254 nm), which forms predominantly CPDs (70 -80%) and 6 -4 dimers (20 -30%), have indicated that mutations and chromosomal aberrations are induced when human cells attempt to replicate the damaged DNA (5, 6). Therefore, the mechanisms whereby human cells complete the replication of template strands containing photoproducts are of considerable interest.UVC inhibits DNA replication in diploid human fibroblast strains by a variety of mechanisms, including G 1 arrest (6) and inhibition of replicon initiation in S phase cells (7). These two checkpoint responses appear to be protective processes, providing more time for DNA repair to remove photoproducts before DNA replication. UVC also induces inhibition of DNA synthesis in active replicons (7,8). The latter is thought to reflect, at least in part, the stalling of DNA replication forks at pyrimidine dimers (9 -11), perhaps due to a reduced capacity of DNA polymerases to incorporate DNA precursors into nascent strands opposite template lesions (12-15). This inhibition, however, is not absolute, and bypass replication eventually takes place, as evidenced by the generation of replicated DNA containing photoproducts and the induction of point mutations at dipyrimidine sites. UV-induced mutations in the p53 tumor suppressor gene in non-melanoma skin cancers are characterized by a high proportion of C 3 T transitions (16 -18). Base substitution mutations at thymines do not occur with high frequency in UV-damaged genes that are replicated at their natural chromosomal locations (19) or as part of shuttle vectors (20 -22).Previous studies have demonstrated that protein extracts from HeLa cells are capable of replicating past CPDs during in vitro replication of UV-damaged plasmids carrying the SV40 origin of replication (23)(24)(25)(26). Experimental evidence in support of this conclusion was found primarily by probing for the presence of sites sensitive to nicking by the CPD-specific enzyme, T4 endonuclease V (T4 endoV), in replicated (DpnI-resistant), closed circular DNA molecules (23)(24)(25)(26). In addition, UV-induced mutagenesis at dipyrimidine sites of randomly damaged plasmids (almost exclusively C 3 T) presumably reflected error-prone bypass replication (trans-lesion synthesis) of CPDs (23,24).Bypass replication of a single dimer strategically placed on one or the other anti-parallel strand of DNA has also been examined (25,26). Inference as to whether bypass replication occurred via leading or lagging strand synthesis was made on the basis of the location and orientation of the dimer, vis à vis...

Interferon-␣ (IFN-␣) is indicated for the treatment of certain viral infections including hepatitis B and C, and cancers such as melanoma. The short circulating half-life of unmodified IFN-␣ makes frequent dosing (daily or three times weekly) over an extended period (6 -12 months or more) necessary. To improve the pharmacokinetics of IFN-␣ and decrease dosing frequency, IFN-␣ was fused to human serum albumin producing a new protein, Albuferon. In vitro comparisons of Albuferon and IFN-␣ showed similar antiviral and antiproliferative activities, although Albuferon was less potent on a molar basis than IFN-␣. Pharmacokinetic and pharmacodynamic properties of the fusion protein were enhanced in monkeys. After a single intravenous injection (30 g/kg,) clearance was 0.9 ml/h/kg, and the terminal half-life was 68 h. After 30 g/kg subcutaneous injection, apparent clearance (clearance divided by bioavailability) was 1.4 ml/h/kg, the terminal half-life was 93 h, and bioavailability was 64%. The rate of clearance of Albuferon was approximately 140-fold slower, and the half-life 18-fold longer, than for IFN-␣ given by the subcutaneous route in other monkey studies. Sera from Albuferon-treated monkeys demonstrated doserelated antiviral activity for Ն8 days based on an in vitro bioassay, whereas antiviral activity from IFN-␣-treated animals was only slightly elevated relative to vehicle on day 0. Significant increases in 2Ј,5Ј-oligoadenylate synthetase mRNA relative to IFN-␣-or vehicle-treated animals were maintained for Ն10 days after subcutaneous dosing. The improved pharmacokinetics of Albuferon are accompanied by an improved pharmacodynamic response suggesting that Albuferon may offer the benefits of less frequent dosing and a potentially improved efficacy profile compared with IFN-␣.

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

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IFN-κ is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-κ on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-κ or two recombinant IFN subtypes, IFN-β and IFN-α2a. Although IFN-α2a failed to stimulate monocyte cytokine secretion, IFN-κ, like IFN-β, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-κ was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-β, IFN-κ did not induce release of IFN-γ by PBL. Expression of the IFN-κ mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-γ stimulation in monocytes, while IFN-β mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-κ was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-κ, like IFN-β, binds strongly to heparin (Kd: 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-κ in monocytes, coupled with the unique induction of IFN-κ mRNA by IFN-γ, indicates a potential role for IFN-κ in the regulation of immune cell functions.

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