Murine xenograft model demonstrates significant radio-sensitising effect of liposomal doxorubicin in a combination therapy for Feline Injection Site Sarcoma.

Therapy of cats suffering from feline injection site sarcomas (FISS) is still a challenging problem, as the recurrence rate after surgery is up to 70%. Four FISS-derived primary tumour cell lines and corresponding xenograft tumour mouse models were established to evaluate the efficacy of a concomitant chemo-/radiation therapy with doxorubicin. In vitro, strongly depending upon the timing of administration, pre-treatment with 0.25 µmol doxorubicin resulted in a significant enhancement of radiation-induced (3.5 Gy) tumour cell death. This result was confirmed in vivo, where only the combined chemo-/radiation therapy resulted in a significant reduction in tumour growth compared to the respective mono-therapies with either doxorubicin or radiation. These results support the use of the concomitant chemo-/radiation therapy for adjuvant treatment of FISS, particularly in advanced or recurrent disease where surgery alone is no longer feasible.

Primary effusion lymphoma cells undergoing human herpesvirus type 8 productive infection produce C-type retroviral particles.

Primary effusion lymphomas (PELs) are invariably infected by the human herpesvirus 8 (HHV8)that is present in most PEL cells as latent virus but replicates in a subset of permissive cells to produce infectious progeny. Here we show that productively infected PEL cells release C-type retrovirus-like particles encoding an Mn++-dependent RT activity, which is typical of endogenous retroviruses. Strikingly, C-type particles are produced only in cells showing advanced HHV8 morphogenesis. Phorbol esters, which induce productive HHV8 replication and morphogenesis in PEL cells, increase RLP production. Phosphonoacetic acid, a blocker of HHV8 late gene expression, inhibits the production of C-type particles, whereas neutralizing anti-alphaIFN antibodies, which are known to increase HHV8 assembly, increases C-type particle production. These data suggest that factors expressed in advanced stages of HHV8 reactivation support endogenous C-type particle morphogenesis in PEL cells.

Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors.

BACKGROUND: A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles. METHODS: In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)-based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3' LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5' LTR of the vector. RESULTS: PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence-activated cell sorting (FACS). After detection of low-level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3' LTR at the R/U5 border to prevent accidental read-through transcription from neighbouring cellular promoters. Virus-containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells. CONCLUSIONS: This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell.

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines.

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi's sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases.

HIV type 1 Nef promotes neoplastic transformation of immortalized neural cells.

To study the effects of HIV-1 Nef on CNS-derived cells in vivo, an expression system based on the murine neural stem cell line C17.2 was established. Stable expression of LAV-1(Bru)-nef in these cells induced a transformed phenotype and enhanced cell growth in soft agar. Further experiments using previously established nef-expressing human astrocytoma cell lines as well as nef-expressing murine fibroblasts suggested a brain cell-specific transforming activity of Nef. After implantation into syngeneic or nude mice both murine and human nef-expressing CNS-derived cells induced tumor development. Interestingly, human astrocytoma cells expressing a Nef mutant carrying a disrupted SH3-binding motif involved in protein-protein interactions failed to induce tumor formation. These in vivo data suggest that Nef promotes neoplastic transformation of immortalized murine neural stem cells and enhances malignancy of low-tumorigenic human astrocytoma cells. Nef may therefore be involved in the development of AIDS-associated brain tumors.

Cell type-specific expression and promoter activity of human endogenous retroviral long terminal repeats.

Evolution over millions of years has adapted several thousand copies of retrovirus-like elements and over 10 times as many solitary long terminal repeats (LTRs) to their present location in the human genome. Transcription of these human endogenous retroviruses (HERVs) has been detected in various cells and tissues, and in some cases their transcriptional control elements have been recruited by cellular genes. We used a retroviral pol-specific expression array to obtain a HERV transcription profile in a variety of human cells such as epidermal keratinocytes, liver cells, kidney cells, pancreatic cells, lymphocytes, and lung fibroblasts. This rapid screening test revealed a distinct HERV pol-expression pattern in each cell type tested so far. About 40 different U3/R regulatory sequences from the HERV-H and HERV-W families were then amplified from actively transcribed 3'HERV LTRs of various cell lines and tissues. Their promoter activities were compared with LTR sequences of other known HERV families in 12 human cell lines using a transient luciferase reporter system. Expression of the isolated HERV LTRs varied significantly in these cell lines, in some cases showing strict cell type specificity. These results suggest that endogenous retroviral LTRs may be a valuable source of transcriptional regulatory elements for the construction of targeted retroviral expression vectors.

Rapid identification of all known retroviral reverse transcriptase sequences with a novel versatile detection assay.

We have developed a highly sensitive, universal assay that allows detection as well as identification of all known retroviral reverse transcriptase (RT)-related nucleic acids in a biological sample by a single two-step experiment. The assay combines polymerase chain reaction (PCR) and reverse dot-blot hybridization (RDBH), using an array of immobilized synthetic retrovirus-specific oligonucleotides and two sets of mixed oligo primers (MOPs). These primers were derived from highly conserved motifs found in all known reverse transcriptase genes. The PCR/RDBH assay was used for qualitative analyses of human endogenous retrovirus (HERV) transcription in peripheral blood mononuclear cells (PBMCs) and in particles released by the human mammary carcinoma-derived cell line T47D. Sensitivity was further demonstrated by detection of down to 10 copies of pig endogenous retrovirus (PERV) DNA in human cDNA samples. Therefore, this assay is particularly useful for the identification of retroviral sequences in xenografts as well as in recipients of xenografted tissues and organs. Moreover, it is a valuable tool to detect retroviral transcripts and particles in cell cultures used for production of therapeutic polypeptides. The assay is further suitable for monitoring vector preparation used in human gene therapy to exclude transfer of copackaged endogenous retroviruses into target cells.

Transcriptional activation of endogenous retroviral sequences in human epidermal keratinocytes by UVB irradiation.

Ultraviolet radiation is a pathogenic factor in various diseases, e. g., autoimmune disorders such as lupus erythematosus. On the other hand, endogenous retroviruses are discussed as etiologic agents in lupus erythematosus. Therefore, we investigated the influence of ultraviolet irradiation on expression of human endogenous retroviral sequences and human endogenous retroviral sequence promoter-driven transcription of cellular genes using human epidermal keratinocytes as a model system. First, conserved sequences of endogenous retroviral pol genes were amplified from cellular mRNA by reverse transcriptase polymerase chain reaction with degenerate oligonucleotide primers. Polymerase chain reaction products were hybridized in a reverse dot blot hybridization assay to a representative number of distinct cloned human endogenous retroviral pol fragments. Using this method, we could show that irradiation with 30 mJ per cm2 ultraviolet B activates transcription of various endogenous retroviral pol sequences in primary epidermal keratinocytes as well as in a spontaneously immortalized keratinocyte cell line (HaCaT). Interestingly, some of these sequences were found to be closely related to pol sequences of human endogenous retroviral sequences which have been shown to be expressed in autoimmune patients. Analysis of human endogenous retroviral pol expression in vivo using skin biopsies of lupus erythematosus patients revealed similar activation patterns. In a second approach, ultraviolet B- induced chimeric transcripts were isolated which are initiated by human endogenous retroviral promoters and proceed into cellular sequences using a newly established modified differential display polymerase chain reaction technique. The activation of human endogenous retroviral sequence transcription by ultraviolet B may contribute to the pathogenesis of lupus erythematosus, where inappropriate antigenic presentation of ultraviolet B-induced viral and cellular proteins could stimulate autoantibody production.

Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi's sarcoma spindle cells.

BACKGROUND: Human herpesvirus 8 (HHV8) infection is associated with all forms of Kaposi's sarcoma (KS). The HHV8 genome locus ORFK13-72-73 (ORF = open reading frame) encodes proteins that may be important in HHV8-mediated pathogenesis, i.e., the latency-associated nuclear antigen (encoded by ORF73), viral-cyc-D (v-cyc-D), a viral homologue of cellular cyclin D (encoded by ORF72), and viral-FLIP (v-FLIP), a homologue of the cellular FLICE (Fas-associated death domain-like interleukin 1 beta-converting enzyme) inhibitory protein (encoded by ORFK13; is an inhibitor of apoptosis [programmed cell death]). Through differential splicing events, this locus expresses individual RNA transcripts that encode all three proteins (tricistronic transcripts) or just two of them (v-FLIP and v-cyc-D; bicistronic transcripts). We examined expression of these transcripts in KS tissues. METHODS: We collected tissues from patients with KS of different stages. By use of an optimized in situ hybridization procedure, we examined different ORFK13-72-73 locus transcripts in HHV8-infected cells in skin lesions and in one adjacent lymph node. Apoptosis in KS lesions was determined by use of an in situ assay. RESULTS AND CONCLUSIONS: Our results indicate the following: 1) Transcripts from the ORFK13-72-73 locus appear to be spliced differentially in latently infected KS cells in skin lesions and in HHV8-infected cells in lymph nodes; specifically, ORFK13-ORF72 bicistronic transcripts were expressed abundantly in KS cells, whereas ORFK13-ORF72-ORF73 tricistronic transcripts were detected only in lymph node cells. 2) Sequences encoding the antiapoptotic protein v-FLIP are expressed at very low levels in early KS lesions, but expression increases dramatically in late-stage lesions. 3) The increase in expression of v-FLIP-encoding transcripts is associated with a reduction in apoptosis in KS lesions. IMPLICATIONS: These data suggest that functional v-FLIP is produced in vivo and that antiapoptotic mechanisms may be involved in the rapid growth of KS lesions, where only a few cells undergoing mitosis are generally observed.

Expression of human herpesvirus-8 (HHV-8) encoded pathogenic genes in Kaposi's sarcoma (KS) primary lesions.

Transcription of six different HHV-8 specific mRNAs was examined in early- and late-stage KS primary lesions. Expression of the latency-associated T0.7 mRNA and of VP23 mRNA which is a specific marker of lytic/productive infection suggested that HHV-8 is secondarily recruited into the KS lesions by productively infected monocytes, macrophages. From these cells HHV-8 is transmitted to the KS spindle cells, which are latently infected. v-BCL-2, v-MCP-1 and v-IL-6 were not expressed in latently infected KS spindle cells, therefore the impact of these factors in KS pathogenesis appears to be low. By contrast, v-Cyclin D was highly expressed in almost all latently infected spindle cells and may therefore be an important factor triggering progression of late-stage KS lesions.

Infection with human immunodeficiency virus-1 increases expression of vascular endothelial cell growth factor in T cells: implications for acquired immunodeficiency syndrome-associated vasculopathy.

Alterations in the vascular system and the onset of angioproliferative lesions such as Kaposi's sarcoma (KS) are common traits of human immunodeficiency virus-1 (HIV-1)-infected patients. To investigate possible factors involved in acquired immunodeficiency syndrome (AIDS)-associated vasculopathy and vascular malfunction, expression of vascular endothelial cell growth factor-A (VEGF-A) was analyzed in HUT 78 T lymphocytes upon infection with HIV-1. VEGF-A was found to be increased in supernatants from infected cells as compared with uninfected cells. In addition, VEGF-A mRNA expression and protein secretion were significantly increased in HUT 78 cells incubated with conditioned medium (CM) derived from HIV-1 chronically infected HUT 78 cells (HIV-TCM) as compared with CM from uninfected cells (TCM). Increase of VEGF-A production in T cells was promoted by inflammatory cytokines (IC) present in HIV-TCM, including tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), interleukin-1beta (IL-1beta), and IL-6. These IC that have been shown to be increased in sera of HIV-1-infected patients and to be increased by HIV-1 infection or cell activation in these individuals as well as HIV-TCM also increased VEGF-A expression in primary T lymphocytes. Consistent with this, VEGF-A concentrations were found to be higher in sera of HIV-1-infected patients with (mean, 357.1 +/- 197.9 pg/mL) and without KS (mean, 256.7 +/- 137.5 pg/mL) as compared with uninfected individuals (mean, 188.6 +/- 91.7 pg/mL). These data suggest that increased secretion of VEGF-A by T lymphocytes of HIV-1-infected individuals may induce vascular leakage and stimulate proliferation of vascular endothelial cells, which are hallmarks of AIDS-associated vasculopathy and especially of KS development.

An antiviral mechanism of nitric oxide: inhibition of a viral protease.

Although nitric oxide (NO) kills or inhibits the replication of a variety of intracellular pathogens, the antimicrobial mechanisms of NO are unknown. Here, we identify a viral protease as a target of NO. The life cycle of many viruses depends upon viral proteases that cleave viral polyproteins into individual polypeptides. NO inactivates the Coxsackievirus protease 3C, an enzyme necessary for the replication of Coxsackievirus. NO S-nitrosylates the cysteine residue in the active site of protease 3C, inhibiting protease activity and interrupting the viral life cycle. Substituting a serine residue for the active site cysteine renders protease 3C resistant to NO inhibition. Since cysteine proteases are critical for virulence or replication of many viruses, bacteria, and parasites, S-nitrosylation of pathogen cysteine proteases may be a general mechanism of antimicrobial host defenses.

HSP47, a collagen-specific molecular chaperone, delays the secretion of type III procollagen transfected in human embryonic kidney cell line 293: a possible role for HSP47 in collagen modification.

HSP47 is a stress protein (heat shock protein) which resides in the endoplasmic reticulum, and is postulated to function as a collagen-specific molecular chaperone. To elucidate the role of HSP47 in procollagen biosynthesis, we have established human embryonic kidney 293 cell lines, which were stably transfected with alpha1(III) procollagen chains with or without HSP47. 293 cells do not produce any extracellular matrix proteins including collagens, and the level of HSP47 expression is almost undetectable in this cell line. Recombinant type III procollagens in 293 cells form trypsin-resistant homotrimers, which are secreted into the medium as trimers in the presence or absence of recombinant mouse HSP47. The secretion of procollagen III was delayed in 293 cells stably transfected with proalpha1(III) collagen chains [293+proalpha1(III) cells] in comparison with human rhabdomyosarcoma cell line RD, which normally produces type III procollagens. In this study, we examined the rate of type III procollagen secretion in detail. In cells cotransfected with mouse HSP47 [293+proalpha1(III)+HSP47 cells], the rate of type III procollagen secretion was slower than in 293+proalpha1(III) cells. The binding of HSP47 with proalpha1(III) collagen chains was confirmed by immunoprecipitation using the chemical cross-linker, DSP. The electrophoretic mobility of proalpha1(III) collagen chains in 293+proalpha1(III) cells was slightly slower than that in RD cells, whereas the recombinant proalpha1(III) chains of 293+proalpha1(III)+HSP47 cells showed almost the same electrophoretic mobility as those of RD cells. The melting temperature (Tm) of type III procollagen in 293+proalpha1(III)+HSP47 cells was almost the same as that in RD cells, and the Tm in 293+proalpha1(III) cells was slightly higher than that in RD cells. These data suggest that the recombinant proalpha1(III) collagen chain is overmodified in 293+proalpha1(III) cells, but not in 293+proalpha1(III)+HSP47 cells.

UV-B irradiated cell lines execute programmed cell death in various forms.

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.

Expression of HHV-8 latency-associated T0.7 RNA in spindle cells and endothelial cells of AIDS-associated, classical and African Kaposi's sarcoma.

Analysis by polymerase chain reaction (PCR) and serological studies have demonstrated a close association between the novel human herpes virus, Kaposi's sarcoma-associated herpes virus (KSHV) or human herpes virus-8 (HHV-8) and the development of Kaposi's sarcoma (KS). To clarify the role of HHV-8 in KS pathogenesis, we investigated at the cellular level by in situ hybridization the expression of a recently described 0.7-kb HHV-8-encoded mRNA (T0.7 mRNA) in KS tissues of different epidemiological origin (AIDS-KS, African endemic KS and classical KS). The T0.7 mRNA likely encodes a small membrane protein, supposedly expressed in latently HHV-8-infected cells. Indeed, we detected T0.7 mRNA in virtually all cells of the cell line BCBL-1 established from a body cavity-based lymphoma (BCBL) and latently infected with HHV-8. In all KS biopsies examined, independent of their epidemiological type, the late-stage (nodular) KS tissues showed a high level of T0.7 mRNA expression in typical KS spindle cells but also in endothelial cells lining blood vessels, indicating latent HHV-8 infection of these cells. The presence of T0.7-expressing cells was restricted to KS tumor tissue and therefore appears to indicate an important role of latent HHV-8 infection in KS pathogenesis.

Structure of a novel extracellular Ca(2+)-binding module in BM-40.

The EF-hand is a highly conserved Ca(2+)-binding motif found in many cytosolic Ca(2+)-modulated proteins. Here we report the crystal structure at 2.0 A resolution of the carboxy-terminal domain of human BM-40 (SPARC, osteonectin), an extracellular matrix protein containing an EF-hand pair. The two EF-hands interact canonically but their detailed structures are unusual. In the first EF-hand a one-residue insertion is accommodated by a cis-peptide bond and by substituting a carboxylate by a peptide carbonyl as a Ca2+ ligand. The second EF-hand is stabilized by a disulphide bond. The EF-hand pair interacts tightly with an amphiphilic amino-terminal helix, reminiscent of target peptide binding by calmodulin. The present structure defines a novel protein module occurring in several other extracellular proteins.

Biological significance of human endogenous retroviral sequences.

Endogenous retroviruses (ERVs) have been known for many years to exist in numerous natural and laboratory animal species. In humans it has been demonstrated that at least 1% of the genome consists of retrovirus-related sequences. Involvement of ERVs in the development of neoplastic and autoimmune diseases in the mouse model implicated a potentially pathogenic role of ERVs for humans, too. The research in this field led to a number of results strongly suggesting that human endogenous retroviral sequences (HERVs) are biologically active, on the RNA and even on the protein level. Particle formation, regulation or dysregulation of cellular gene expression, and synthesis of potentially pathogenic viral proteins indicate the broad spectrum of mechanisms by which HERVs may obtain biological significance.

The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV.

The extracellular glycoprotein BM-40 consists of three domains, an acidic domain I, a follistatin (FS)-like domain II and a calcium-binding EC domain with an EF-hand related motif. BM-40 and several other related proteins (QR1, SC1/hevin, testican and tsc-36/FRP) are members of a novel modular protein family that share the FS domain followed by an EC domain. We have expressed this pair of FS and EC domains (mutant delta I) and the calcium-binding EC domain alone (mutant delta I, II) of human BM-40 as recombinant proteins in human 293 cells. Circular dichroism demonstrated that both mutants were obtained as folded proteins with a distinct three-dimensional conformation. In addition, mutant delta I, II could be readily crystallized and diffraction patterns with a resolution limit of 2.4 A resolution were obtained. Calcium binding to this fragment was ten times weaker (Kd = 0.8 microM) than for the wild-type protein. Identical reversible increases in alpha-helicity upon calcium binding were observed for the 150-residue long mutant delta I, II and for BM-40 (286 residues). A 26-residue synthetic peptide corresponding to the EF-hand related motif exhibited much weaker calcium binding. The apparent dissociation constant decreased with increasing peptide concentration (from Kd 2.4 mM at 1 microM, to Kd 0.3 mM at 100 microM peptide concentration) and calcium binding was accompanied by dimerization of the peptide. This suggests that for strong calcium binding the EF-hand related motif has to be embedded into a larger protein domain that can form an autonomously folding protein module. The EC domain was also shown by surface plasmon resonance assay to be responsible for calcium-dependent binding to collagen IV with an affinity (Kd = 19 microM) only sixfold lower than that of intact human BM-40.

Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification.

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase-catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 were identified as major amine acceptor sites. This was confirmed with several mutant proteins, including deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N-terminal sequence (APQQEAL) to an unrelated protein. The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level. For high efficiency amine incorporation an intact domain I is required. The conservation of at least one of the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates.