Tomato thymidine kinase-based suicide gene therapy for malignant glioma--an alternative for Herpes Simplex virus-1 thymidine kinase.

Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Comparison on collagen gene expression in the developing chick embryo tendon and heart. Tissue and development time-dependent action of dexamethasone.

Glucocorticoids modulate various cellular functions such as proliferation, energy metabolism and the synthesis of proteins. In the present study, the response of collagen genes to dexamethasone in different stages of chick embryo development was studied in tendon and heart using Northern blot analysis and specific cDNA probes. The changes in collagen gene expression were compared to alterations in two reference mRNAs: actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of specific mRNAs measured per ribosomal RNA in tendon and heart varied markedly during normal development. In tendon the relative levels of alpha 1(I), alpha 2(I) and alpha 1(III) collagen mRNAs were highest between days 14-16 when also the synthesis of matrix proteins is most active. In heart the levels of these mRNAs peaked at day 12. In addition, qualitative differences were observed in the expression of actin genes between tendon and heart. Dexamethasone in high dose decreased collagen mRNA levels in tendons, while in heart a stimulatory effect was noted. Dexamethasone also decreased GAPDH mRNA levels in tendons. The alterations in gene expression after dexamethasone treatment in tendon and heart did not correlate with the level of specific glucocorticoid receptors, which varied markedly during the development of chick embryos. The cDNA for pro alpha 1(I) collagen hybridized to two transcripts corresponding to 6.2 and 5.1 kb in tendon and heart. During normal development of chick embryos the ratio of 6.2/5.1 kb mRNAs decreased markedly in heart, but no such change was observed in tendons. Dexamethasone, however, decreased the ratio of 6.2/5.1 kb transcripts in tendons. There was a significant correlation between the ratio 6.2/5.1 kb transcripts and total alpha 1(I) mRNA both in tendon and heart, suggesting that the 6.2 kb transcript may be associated with the rate of synthesis of type I collagen.

Expression of osteonectin, decorin, and transforming growth factor-beta 1 genes in fibroblasts cultured from patients with systemic sclerosis and morphea.

A characteristic feature of fibroblasts cultured from affected skin areas of patients with systemic sclerosis (SSc) and localized scleroderma (morphea) is excessive activation of collagen biosynthesis. To elucidate the nature of fibroblast activation in scleroderma we have studied the expression of 3 noncollagenous connective tissue components, osteonectin, small dermatan sulfate proteoglycan (proteoglycan II, decorin), and transforming growth factor-beta 1 (TGF-beta 1), by measuring their mRNA levels in fibroblast cultures from 6 patients with SSc and 3 with morphea. A clear correlation was observed between the increase in type I collagen and osteonectin mRNA in these cell lines. The apparent overproduction of osteonectin by scleroderma fibroblasts is in accordance with the suggested activation of osteonectin expression during tissue remodeling. The levels of decorin mRNA showed marked variation in the cell lines, but were in no correlation with collagen or osteonectin mRNA. The levels of TGF-beta 1 mRNA were found to be slightly elevated in fibroblasts grown from affected scleroderma skin. This may suggest that this potent activator of collagen production has a role during the initial activation of dermal fibroblasts both in SSc and morphea.

Purification and molecular cloning of the "A" chain of a rat heteromeric CCAAT-binding protein. Sequence identity with the yeast HAP3 transcription factor.

CCAAT-binding factor (CBF) is a heteromeric mammalian transcription factor which binds to sequences containing a CCAAT motif in a number of promoters such as those for type I collagen, albumin, MHC Class II, beta-actin, and others. It consists of two different components that are both needed for DNA binding. We have purified the "A" chain of CBF to apparent homogeneity by sequence-specific DNA affinity chromatography followed by Mono S and Mono Q ion-exchange chromatography and obtained the amino acid sequences of tryptic peptides of this polypeptide. Amino acid sequences of two of these tryptic peptides were used to synthesize oligonucleotide primers. The primers served to obtain a small cDNA by the polymerase chain reaction method, which was then further used to obtain larger cDNA clones. DNA sequence analysis of a representative cDNA clone revealed the presence of an open reading frame of 207 amino acids coding for a putative polypeptide of 25 kDa. Transcription of these cDNAs in vitro followed by translation in a reticulocyte lysate produced a polypeptide that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility as the native A chain. The deduced amino acid sequence of the A chain showed a remarkable identity over a length of 90-amino acid residues with a sequence of the Hap3 polypeptide, a component of a heteromeric multisubunit yeast transcription factor.

The B subunit of a rat heteromeric CCAAT-binding transcription factor shows a striking sequence identity with the yeast Hap2 transcription factor.

CBF is a heteromeric mammalian transcription factor that binds to CCAAT sequences in a number of promoters such as the two type I collagen promoters, the albumin promoter, the major histocompatibility complex class II promoter, and others. It is composed of two components, A and B, that are both needed for DNA binding. We have isolated a rat cDNA containing the complete 341-amino acid coding sequence of the B component of CBF. Expression of this cDNA in vitro generates a polypeptide that shows the same dependency on the A component as the native B component in the formation of a complex with a CCAAT-containing DNA. The C-terminal portion of the B component from residue 260 to residue 312 shows a 75% sequence identity with a portion of the Hap2 protein, a component of a heteromeric CCAAT-binding protein in yeast. In contrast, the rest of the protein shows little sequence homology with Hap2, although both proteins contain glutamine-rich domains. In the B component of CBF this domain spans the amino-terminal 60% of the protein, whereas in Hap2 this domain is much smaller. Hence, only a few changes in one domain of this protein were tolerated during evolution between yeast and mammals, whereas the rest of the protein diverged much more extensively.

Growth-dependent modulation of type I collagen production and mRNA levels in cultured human skin fibroblasts.

Five human skin fibroblast lines were studied for type I collagen production and type I procollagen mRNA levels through the different growth phases. The cells were plated at low density and followed for 11 days at daily intervals through the stages of rapid growth and visual confluency until the cultures reached stationary growth phase. Each day one culture flask was labeled with [3H]proline for 24 h, and analyzed for production of radiolabeled type I collagen into culture medium. The cell layers were counted and subjected to isolation of cytoplasmic RNA and determination of type I procollagen mRNA levels. The results revealed an approx. 2-fold increase in procollagen production and mRNA levels when the cells reached visual confluency. Thereafter the synthesis rates and mRNA levels remained relatively constant, although a decreasing tendency of both parameters was observed upon further culturing. The results confirm that determination of cell density is important when cell cultures are used for measurement of collagen synthesis or mRNA levels. For determination of pro alpha 2(I) collagen mRNA an 1193 bp cDNA clone was constructed using RNA extracted from human fetal calvaria. Sequencing of the clone revealed some nucleotide and amino acid differences between the previously published sequences. This suggests the presence of more individual variation in procollagen coding sequences than expected.

Control of type I collagen genes in scleroderma and normal fibroblasts.

Increased gene transcription is a mechanism responsible for the exaggerated accumulation of type I and III collagens in scleroderma and other fibrotic syndromes. We review recent studies on the mechanisms that control transcription of the alpha 1 (I) and alpha 2 (I) collagen genes. Several specific DNA binding proteins have been identified which either activate or inhibit transcription of these genes. It is probable that the complexity of transcription factors that control the type I collagen genes corresponds to a multiplicity of cytokines and other hormonal effectors, which influence the expression of these genes by binding to specific receptors and by activating intracellular signaling pathways.

Characterization of excessive collagen production during development of pulmonary fibrosis induced by chronic silica inhalation in rats.

The activation of collagen synthesis during development of silicotic fibrosis was studied in rats exposed, in dusting chambers, to respirable SiO2 for periods of 2, 4, 6 or 12 months. Control animals were exposed similarly to clean air or TiO2. Development of fibrosis was followed by histological examination, measurement of lung weight and determination of lung collagen content (as hydroxyproline). A steady increase in lung weight and collagen content together with changes in cellularity and metabolic activity of the lungs, as ascertained by chemical determination of DNA and RNA, were measured in the lungs of the SiO2-exposed animals. Hybridization of total lung RNA, extracted at each time point, with cDNA probes specific for type I and type III procollagen mRNA levels showed that the development of fibrosis was associated with increased levels, as compared to age matched controls, of pulmonary procollagen mRNAs. Interestingly, the highest levels of procollagen mRNAs were observed in young (pretreatment control) animals, suggesting that during pulmonary development collagen metabolism in lungs is even greater than during development of fibrosis. In rats exposed to SiO2 the increase in type III procollagen mRNA occurred earlier than the increase in type I procollagen mRNAs. These observations demonstrate both age-dependent and silicosis-related changes in pulmonary procollagen mRNA levels. The results suggest that development of silicosis is associated with an altered capacity of the lungs to regulate collagen accumulation.

Construction of a human pro alpha 1(III) collagen cDNA clone and localization of type III collagen expression in human fetal tissues.

A cDNA clone for human pro alpha 1(III) collagen mRNA was isolated from a cDNA library constructed for human fetal skin RNA. The clone, pHFS3, was identified by restriction mapping and sequencing. Comparison with previously published human type III collagen sequences revealed some differences which may reflect individual variation. The clone was used to study the expression of type III collagen mRNA in various fetal tissues in comparison to the expression of type I collagen mRNAs. In 15-18-week fetal skin the ratio of alpha 1(I) to alpha 1(III) collagen mRNAs was 0.8. Diaphyseal and calvarial bone contained high amounts of type I collagen mRNA and low levels of type III collagen mRNA, resulting in high type I/type III ratios. In situ hybridization of sections of skeletal tissues was employed to identify the cells containing the mRNAs for types I, II and III procollagens. The results revealed differential expression patterns for these three collagen types in various human fetal tissues. Lack of coordinate expression suggests that production of type I and type III collagens is under different regulatory mechanisms in developing skeletal tissues.

Comparison of the effects of dexamethasone and 13-cis-retinoic acid on connective tissue biosynthesis in human skin fibroblasts.

The effects of glucocorticoids and retinoids on connective tissue biosynthesis were studied in cultured human skin fibroblasts (HSFs). More specifically attention was paid to the effects of dexamethasone and 13-cis-retinoic acid (RA) on total protein and collagen synthesis and on collagen and fibronectin mRNA levels. The results indicated that dexamethasone reduced the relative collagen synthesis and collagen mRNA levels in HSFs and increased the total incorporation of proline into proteins, the latter effect being due to increased activity in the intracellular proline pool. 13-cis-RA did not affect collagen synthesis at the concentration studied (10(-7) M) but it did reduce the corresponding mRNA levels. Simultaneous addition of both dexamethasone and 13-cis-RA or etretinate resulted in the largest decrease in type I and type III procollagen mRNA levels, indicating that retinoids do not oppose the effect of glucocorticoids on collagen synthesis in cultured HSFs. For comparison the effects of dexamethasone and 13-cis-RA on the mRNA levels of another extracellular matrix component, fibronectin, and of a constitutive enzyme, glyceraldehyde-3-phosphate dehydrogenase, were also studied. The results indicated, that dexamethasone treatment did not alter fibronectin mRNA levels in HSFs, while 13-cis-RA did so to a marked extent. Both dexamethasone and 13-cis-RA also reduced the mRNA level of glyceraldehyde-3-phosphate dehydrogenase, indicating that glucocorticoids and retinoids have both similar and different effects on gene expression in HSF.

13-cis retinoic acid and dexamethasone modulate the gene expression of epidermal growth factor receptor and fibroblast proteoglycan 40 core protein in human skin fibroblasts.

The effects of 13-cis retinoic acid (RA) and dexamethasone on the levels of epidermal growth factor (EGF) receptor and fibroblast derived proteoglycan core protein (PG40) mRNAs were studied in human skin fibroblasts. The EGF receptor is involved in the regulation of cellular proliferation and the synthesis of matrix proteins, and proteoglycan 40 is important for cell attachment and interaction with collagen and fibronectin. 13-cis-RA at a concentration of 10(-7) M markedly reduced the levels of the EGF receptor and PG40 mRNAs, the decreases being 33 and 56%, respectively. Dexamethasone reduced these mRNAs markedly less. Simultaneous treatment of the fibroblasts with 13-cis-RA and dexamethasone resulted in similar decreases in EGF receptor and PG40 mRNAs as with 13-cis-RA alone. Surprisingly, the proliferation rate of the fibroblasts was increased in the presence of dexamethasone under conditions similar to those which caused slight decrease in the EGF receptor mRNA levels. This indicates that glucocorticoids also affect the cellular growth by mechanisms which do not involve EGF receptors.

Expression of the c-Ha-ras and neu oncogenes in DMBA-induced, anti-estrogen-treated rat mammary tumors.

Dimethylbenzanthracene (DMBA)-induced rat mammary tumors were analyzed for the structure and expression of the oncogenes c-Ha-ras and neu and the effects of anti-estrogen treatment. Tumor samples were divided into 3 groups, the first consisting of untreated tumors, the second of anti-estrogen (toremifene)-treated unresponsive (growing) tumors, and the third of toremifene-treated responsive (regressing) tumors. DNA and RNA derived from normal tissues of the same experimental animals were also analyzed. In Southern blot analysis of genomic DNAs, 2 tumors out of 23 contained a new Xbal site in the Ha-ras gene, indicating a point mutation in the second nucleotide of codon 61. Both of these tumors belonged to the group that had not received toremifene. No amplifications of the Ha-ras or the neu genes were observed. Although greatly variable, the levels of Ha-ras mRNA were highest in untreated tumors, lower in toremifene-treated, unresponsive tumors and even lower in toremifene-treated, regressing tumors, corresponding approximately to the levels detected in normal liver and uterus of untreated animals. Expression of the neu mRNA was variable and considerably lower than that of Ha-ras mRNA. It was similar in all 3 groups and somewhat elevated than in several non-malignant control tissues. Localization of c-Ha-ras expression by in situ hybridization revealed a relatively even distribution of the mRNA throughout the mammary tissue. The results suggest that mechanisms other than activation of the c-Ha-ras or neu genes are important for progression and regression of DMBA-induced rat mammary carcinomas.

Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization.

Skin biopsies from seven patients with localized scleroderma (morphea) and from two healthy individuals were studied by in situ hybridization to localize the cells responsible for increased procollagen production. In scleroderma lesions, high levels of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNAs were detected in some but not all fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens hybridized at the same time under identical conditions. Most of the scleroderma samples represented intermediate stages where the fibroblasts containing elevated levels of type I and type III procollagen mRNAs were located in the papillary and upper reticular layer of the dermis. One of the scleroderma samples from an early inflammatory stage of the disease was found to contain activated fibroblasts in all dermal layers and also in aggregates adjacent to inflammatory cell infiltrates. In situ analyses were also performed on cell cultures from affected and unaffected skin of one scleroderma patient. These experiments revealed a homogeneous population of activated fibroblasts in cultures producing high levels of collagen. The results suggest that development of fibrosis in scleroderma could evolve through activation of a certain fibroblast subpopulation. During cell culturing, however, cell selection or uncharacterized regulatory mechanisms appear to modulate the behavior of these cells with respect to collagen production.

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones.

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.

Interferon-alpha and interferon-gamma reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture.

The effects of interferon-alpha and interferon-gamma on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-gamma treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-gamma reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-alpha on procollagen mRNA levels was somewhat weaker than that of interferon-gamma. The results suggest potential use of interferon-gamma in treatment and prevention of human fibrotic conditions.

Determination of the single polyadenylation site of the human pro alpha 1(II) collagen gene.

Several cDNA clones for the human type II procollagen mRNA were isolated from a cartilage cDNA library. Six of the clones containing the longest inserts were subjected to restriction site mapping for alignment. All these six clones extended to the poly A tail. The longest clone, containing a 1470 bp insert, was named pHCAR3. Sequencing of pHCAR3 made it clear that neither of the two canonical AATAAA sequences of the human type II collagen gene is used as the polyadenylation signal. Two 60 bp stretches of high interspecies homology terminating in a hexanucleotide ATTAAA, located 23 nucleotides upstream of the poly A tail, apparently have an important role in determining the single polyadenylation signal for this gene. S1 protection experiments confirmed these observations.