Association between recent herpes zoster but not herpes simplex infection and subsequent risk of malignancy in women: a retrospective cohort study.

The association between herpes zoster and subsequent cancer risk is still unclear. Consequently, doubts remain regarding the need for investigation of herpes patients for co-existing or subsequent malignancy. This is a retrospective cohort study comparing cancer risk in patients after herpes zoster and age-/sex-matched non-herpes zoster patients, in a primary care-based continuous morbidity database. We tested for interaction by gender, age, diabetes, HRT use or antiviral therapy. Analyses were repeated for patients with and without herpes simplex. The hazard ratio (HR) comparing cancer risk in herpes zoster vs. control patients was significant in all women, women aged > 65 years and subgroups of breast and colorectal cancer (HRs 1·60, 1·82, 2·14, 2·19, respectively). For men, a significant association was found for haematological cancers (HR 2·92). No associations were found with herpes simplex. No interaction was identified with antiviral therapy, diabetes or HRT treatment. We concluded that there was a moderate significant association between herpes zoster and subsequent cancer risk in women aged > 65 years, without any influence of antiviral therapy. No association was found with herpes simplex. There is insufficient reason for extensively testing older patients with herpes zoster or herpes simplex for the presence of occult cancer.

In vitro-selected drug-resistant varicella-zoster virus mutants in the thymidine kinase and DNA polymerase genes yield novel phenotype-genotype associations and highlight differences between antiherpesvirus drugs.

Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACV(r)) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the "palm domain" of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Malaria: host-pathogen interactions, immunopathological complications and therapy.

Malaria is a global tropical disease causing more than 1 million deaths and 300 million clinical cases every year. It is caused by parasites from the genus Plasmodium and is transmitted by Anopheles mosquitoes. Approximately 3 billion people live in malaria-endemic regions and a majority of them are infected. In this review, we discuss the life cycle of the parasite, the complex interactions with the human host and the ensuing immune reactions and complications. The immune system plays a dual role in malaria, by providing life-saving immunity against the parasite, but also by causing often lethal complications in a number of patients. Cytokines, chemokines and proteases are key players in the immunopathological complications, and we propose immunomodulation with dexamethasone as a promising strategy for the therapy of malaria-associated acute respiratory distress syndrome.

A novel, NH2-terminal sequence-characterized human monokine possessing neutrophil chemotactic, skin-reactive, and granulocytosis-promoting activity.

A factor able to induce an early local inflammation in rabbit skin was detected in the supernatant of mitogen-stimulated human blood leukocytes. The factor was different from IL-1 which, although present in the supernatants, was chemically separable from the factor and induced a late rather than an early skin response. Other biological effects of the principal factor were its in vitro chemotactic effects on granulocytes and its ability to induce rapid granulocytosis upon intravenous injection in rabbits. When tested under the same conditions, IL-1 beta did not act chemotactically and induced granulocytosis at a later time. The factor was purified to homogeneity and identified by electrophoretic mobility as a protein of Mr 6,500. Amino acid sequence analysis revealed the presence of an uncontaminated NH2-terminal sequence identical to a segment of the sequence previously predicted from the cDNA clone (3-10C) copied from an mRNA isolated from human leukocytes and coding for a protein of unknown function. The NH2-terminal sequence of the factor also showed extensive homology to that of the platelet factors beta-thromboglobulin (beta TG) and platelet factor 4 (PF-4). Studies done to identify the cell source of the factor revealed that it was produced by adherent mononuclear cells but not by platelets, while the opposite was true for beta TG.

Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family.

Cytokine-stimulated human osteosarcoma cells (MG-63) secrete several related chemotactic factors, including the neutrophil-activating protein interleukin 8 (IL-8) and the monocyte chemotactic protein (MCP)-1. We describe the isolation and characterization of two novel monocyte chemotactic factors from this tumor cell line. Although these proteins copurified with MCP-1 and IL-8 on heparin-Sepharose, they could be separated by cation-exchange fast protein liquid chromatography and reverse-phase high-performance liquid chromatography. The corresponding 7.5- and 11-kD proteins were NH2-terminally blocked but were identified by sequencing peptide fragments. They showed a primary structure mostly related to that of MCP-1 and were therefore designated MCP-2 and MCP-3, respectively. These molecules can be classified in a subfamily of proinflammatory proteins characterized by the conservation of cysteine residues. MCP-2 and MCP-3 are also functionally related to MCP-1 because they specifically attract monocytes, but not neutrophils, in vitro. The chemotactic potency (specific activity) was comparable for all three MCPs. Intradermal injection of these proteins in rabbits resulted in selective monocyte recruitment in vivo. Since tumor cells are good producers of leukocyte chemotactic factors, it could be questioned whether these molecules can indirectly control tumor growth by attracting leukocytes or whether they rather promote invasion by the secretion of proteases from the attracted cells.

Identification of the human 26-kD protein, interferon beta 2 (IFN-beta 2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor.

A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells (MG-63) after treatment with IL-1 or TNF. The hybridoma-plasmacytoma growth factor (HPGF) is identified with a 26 kD protein whose mRNA was previously shown to be induced in the same cells by the same inducers. First, poly(A)-rich RNA extracted from IL-1-treated cells could be enriched in HPGF-mRNA content by hybridization to 26 kD cDNA. Second, MG-63-derived HPGF purified to electrophoretic homogeneity was subjected to amino acid sequence analysis, whereby the NH2-terminal sequence was found to match the nucleotide sequence of a 26 kD cDNA clone.

Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions.

Regulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B-deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B-deficient mice were resistant to this tail pathology. Gelatinase B-deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.

The MCP/eotaxin subfamily of CC chemokines.

Migration of leukocytes from the bone marrow to the circulation, the primary lymphoid organs and inflammatory sites is directed by chemokines and specific receptor interactions. Besides the role of this group of low molecular weight cytokines in leukocyte attraction and activation, anti-HIV and hematopoietic activities were also attributed to chemokines. On the basis of the number and arrangement of the conserved cysteines, chemokines are subdivided in two multi-member families, namely the CXC and CC chemokines, whereas fractalkine (CX3C) and lymphotactin (C) are unique relatives. The CC chemokines possess four cysteines of which the first two are adjacent. Functionally, they form a rather heterogeneous family. Here, the focus is on the monocyte chemotactic proteins and eotaxin which, on a structural basis, can be considered as a CC chemokine subfamily. Not only the protein sequences, but also the gene structures, chromosomal location, biological activities and receptor usage exhibit considerable similarities. The review is complemented with a comparison of the biological functions of the MCP/eotaxin-subfamily in physiology and pathology.

11ß-hydroxysteroid dehydrogenase type 1 has no effect on survival during experimental malaria but affects parasitemia in a parasite strain-specific manner.

Malaria is a global disease associated with considerable mortality and morbidity. An appropriately balanced immune response is crucial in determining the outcome of malarial infection. The glucocorticoid (GC) metabolising enzyme, 11ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1) converts intrinsically inert GCs into active GCs. 11ß-HSD1 shapes endogenous GC action and is immunomodulatory. We investigated the role of 11ß-HSD1 in two mouse models of malaria. 11ß-HSD1 deficiency did not affect survival after malaria infection, but it increased disease severity and parasitemia in mice infected with Plasmodium chabaudi AS. In contrast, 11ß-HSD1 deficiency rather decreased parasitemia in mice infected with the reticulocyte-restricted parasite Plasmodium berghei NK65 1556Cl1. Malaria-induced antibody production and pathology were unaltered by 11ß-HSD1 deficiency though plasma levels of IL-4, IL-6 and TNF-α were slightly affected by 11ß-HSD1 deficiency, dependent on the infecting parasite. These data suggest that 11ß-HSD1 is not crucial for survival of experimental malaria, but alters its progression in a parasite strain-specific manner.

Molecular and genetic studies on the region of translocation and duplication in the neuroblastoma cell line NGP at the 1p36.13-p36.32 chromosomal site.

Cytogenetic and molecular studies suggest that chromosome 1p might contain oncosuppressor genes involved in the pathogenesis of neuroblastoma and other adult tumors. The isolation of these genes by the 'positional cloning' approach will be facilitated by the characterization of cell lines with well defined chromosomal aberrations. In the present report we provide molecular data on the NGP neuroblastoma cell line which contains a reciprocal t(1;15) translocation. Two regions, possibly hosting oncosuppressor genes, have been identified: one is distal to the ENO1 locus, the other one is comprised between PND and A12M2 and corresponds to that of a constitutional t(1;17) translocation described in a neuroblastoma patient. Genetic data also suggest that the NGP cell line, despite the presence of two chromosomes 1, might be hemizygous for the subtelomeric 1p region.

Constitutional translocation t(1;17)(p36.31-p36.13;q11.2-q12.1) in a neuroblastoma patient. Establishment of somatic cell hybrids and identification of PND/A12M2 on chromosome 1 and NF1/SCYA7 on chromosome 17 as breakpoint flanking single copy markers.

Cytogenetic and molecular studies in neuroblastoma suggest the presence of a tumor suppressor gene at the distal band p36 of human chromosome 1. We described a constitutional translocation t(1;17)(p36;q12-q21), involving the critical region 1p36, in a patient with neuroblastoma, and hypothesized that the translocation predisposed the patient to tumor development. Here we report the molecular delineation of the translocation breakpoints. Somatic cell hybrids were generated by fusion of the patient's fibroblasts with the thymidine kinase deficient hamster cell line, a3. In hybrid cell lines which retained the human derivative chromosomes, the position of chromosome 1p and 17q DNA probes respective to the translocation breakpoints was determined by fluorescence in situ hybridization and Southern blot analysis. The chromosome 1p breakpoint was localized within a repetitive region encoding t-RNA genes, with 12A-2 (PND) as most distal and pHE2.6 (A12M2) as most proximal single-copy breakpoint flanking markers. For the chromosome 17 breakpoint, the proximal and distal flanking markers were identified as 7G4 (NF1) and cMCP-3 (SCYA7), respectively. In this study, cMCP-3 (SCYA7), encoding the human monocyte chemotactic protein-3, was mapped between NF1 and ERBB2. As a pivotal step towards breakpoint cloning, at present these flanking markers optimally delineate the breakpoint regions of both chromosomes 1 and 17 at the molecular level.

Synergistic and selective stimulation of gelatinase B production in macrophages by lipopolysaccharide, trans-retinoic acid and CGP 41251, a protein kinase C regulator.

The production of gelatinase B by macrophages is relevant in the immunological and migratory functions of macrophages. CGP 41251, an inhibitor of protein kinase C (PKC), was found to stimulate the expression of gelatinase B in macrophages, as shown by the study of two different monocytic/macrophagic cell lines, mouse RAW 264.7 and human THP-1 cells. When human monocytes and rat peritoneal macrophages were treated with CGP 41251, insignificant increases of 10 and 25% were obtained. This can possibly be due to the presence of contaminating cells in these two enriched populations, since the CGP 41251 treatment of non-macrophagic cell lines inhibited their PMA-induced gelatinase B production. Taken together, these results suggest that the stimulatory effect of CGP 41251 is specific to cells of the monocytic lineage. Using RAW 264.7 cells as a model, the effect of CGP 41251 is additive to that obtained using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), as revealed by gelatin zymography and Northern blot analysis. The stimulatory effect of CGP 41251 on gelatinase B production in RAW 264.7 was: (a) inhibited by calphostin C (as is the LPS-induced response), indicating a PKC-dependence; (b) inhibited by dexamethasone (as opposed to the LPS-induced response); and (c) enhanced by addition of trans-retinoic acid (RA). In fact, RA can induce gelatinase B production, either alone or in synergy with LPS and/or CGP 41251, since the combination of the three agents gives the highest gelatinase B response, at both the protein and the mRNA levels. This represents an important observation considering the RA is now being tested as an anti-cancer agent and proposed for prevention studies.

Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate.

Several human cell lines were studied for the production of gelatinases. Diploid fibroblasts, the melanoma cell line Bowes, the MG-63 osteosarcoma cell line and the human hepatoma cell line Malavu all constitutively produced a 67 kDa gelatinase. Gelatinolytic enzymes were quantified by a sensitive zymographic substrate conversion assay. Upon induction with phorbol 12-myristate 13-acetate (PMA), the human hepatoma cell line secreted considerable amounts of an 85 kDa gelatinase activity. The induction process was time- and dose-dependent. It represented a true increase in production per individual cell and was associated by a marked change of the cell morphology. The effect of various proteinase inhibitors and the maximal activity of the enzyme near neutral pH demonstrate that it is a neutral metalloproteinase. Characterization studies showed the 85 kDa gelatinase to be transformed to lower molecular weight, active forms by treatment with p-aminophenylmercuric acetate (APMA) or trypsin.

Matrix remodelling enzymes, the protease cascade and glycosylation.

Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1).

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes.

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.

The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator.

The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).

Oligosaccharides of recombinant mouse gelatinase B variants.

Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars. Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC. This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants. The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.

Immunogenization of a murine T-cell lymphoma via transfection with interferon-gamma.

Tumor cell variants were derived from the BW5147 T-cell lymphoma that differ in major histocompatibility complex (MHC) class I antigen expression, tumorigenicity and metastatic potential. In general, increased H-2Kk expression was found to be correlated with a reduced tumorigenicity and spontaneous metastasis. CD8+ T cells were identified in the immune recognition of such variants, implicating a role for H-2Kk in the presentation of tumor-associated antigens. In the present study, H-2Kk+ BW variants were transfected with a gene encoding interferon-gamma (IFN-gamma), a potent inducer of MHC class I expression. The resulting transfectants exhibited an increased expression of H-2Kk and concomitantly an inability to generate visible tumors and a reduced metastatic capacity. Furthermore, immunization with the IFN-gamma transfectants resulted in an increased generation of cytotoxic T lymphocytes (CTLs) that lysed both the transfectants and the parental tumor cells. Based on these results, vaccinations with the IFN-gamma transfectants were performed against the parental tumor cells. The results clearly demonstrated that such vaccinations reduced significantly the tumorigenicity and metastatic capacity of the parental tumor cells. Hence, in this tumor model, IFN-gamma gene transfection provides a means to immunogenize H-2Kk+ BW tumor cells.

Transcriptional control of the human MCP-2 gene promoter by IFN-gamma and IL-1beta in connective tissue cells.

Human monocyte chemotactic protein-2 (MCP-2) is a member of the CC chemokine family. It is produced by mononuclear leukocytes, diploid fibroblasts, and tumor cells after induction with IL-1beta or IFN-gamma. To understand the transcriptional regulation of the gene, we have analyzed the structure and function of the promoter region. The sequence of the 5'-flanking region was determined and the transcription start site was found to be located at 68 nucleotides upstream of the ATG translation start codon. 5'-Deletion mutants were generated and transfected into E6SM diploid fibroblasts and MG-63 osteosarcoma cells. Expression was measured by luciferase assay in transfected unstimulated cells and after stimulation with IL-1beta, IFN-gamma, or a combination. The region between nucleotides -143 and -73 (relative to the transcription initiation site), containing putative cis-elements for GATA-1, H-APF1, AP-1, and GAS, is important for basal transcription levels in both cell lines. Stimulation for 18 h with IL-1beta alone failed to affect expression of any of the constructs both in diploid fibroblasts and in osteosarcoma cells. In both cell lines IFN-gamma increased the activity of all mutants that possessed the region between -340 and -301. In MG-63 cells, stimulation with the combination of IL-1beta and IFN-gamma caused an additional increase in expression of the constructs from -340 onward. Finally, the presence of transcription factors in nuclear extracts of MG-63 cells and their specificity to bind to various oligonucleotide probes in this [-340; -301] region were evidenced by electromobility shift assays. These results show that IFN-gamma, produced by lymphocytes and NK cells, induces the transcription of the MCP-2 gene in fibroblasts and thereby can indirectly contribute to recruitment of various leukocyte cell types to inflammatory sites.

Role of C-X-C chemokines as regulators of angiogenesis in lung cancer.

Lung cancer is the leading cause of malignancy-related mortality in the U.S. and is predicted to increase over the remainder of this decade. Despite attempts to advance early diagnosis and use combination therapies, the clinical response of this cancer yields an overall 5-year survival rate of less than 15%. Clearly, new strategies for therapy are indicated. Although carcinogenesis is complex, tumor growth beyond 1-2 mm3 is dependent on angiogenesis. One of the potential mechanisms that allows for tumorigenesis is dysregulation of the balance of angiogenic and angiostatic factors that favors net neovascularization within the primary tumor. Numerous studies have investigated the role of a variety of molecules in the regulation of angiogenesis. Recently, interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be an angiogenic factor. In contrast, platelet factor 4 (PF4), another C-X-C chemokine, has been shown to have angiostatic properties. It is interesting that the major structural difference between IL-8 and PF4 is the presence of the NH2-terminal ELR (Glu-Leu-Arg) motif that precedes the first cysteine amino acid residue of IL-8 and is important in ligand/receptor interactions. We hypothesize that angiogenesis associated with tumorigenesis is dependent on members of the C-X-C chemokine family acting as either angiogenic or angiostatic factors. This paradigm predicts that the biological balance in the expression of these C-X-C chemokines dictates whether the neoplasm grows and develops metastatic potential or regresses. In this review we discuss our recent laboratory findings that support this contention and suggest that further elucidation of the biology of C-X-C chemokines in the context of neovascularization of nonsmall cell lung cancer will permit novel targeted therapy aimed specifically at attenuating tumor growth and metastasis.