Expression of cathepsin L and its endogenous inhibitors in immortal and transformed fibroblasts.

METHODS: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. RESULTS: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. CONCLUSIONS: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.

Cloning of the murine Drm gene (Cktsf1b1) and characterization of its oncogene suppressible promoter.

The Drm gene, first identified in rat cells in our laboratory, appears to play a significant role in early embryo patterning and limb bud development. We have now isolated mouse Drm (mDrm) cDNA as well as genomic DNA clones and have mapped the Drm gene (Cktsf1b1) to murine chromosome 2. Cktsf1b1 is regulated in a tissue specific fashion and is expressed only in nontransformed mouse cells or primary fibroblasts in culture, but not in established transformed or tumor-derived mouse cell lines. The major transcription start sites map to within 69 bp upstream of the initiating ATG. A promoter was contained in the -214 to +1 bp 5' flanking region, and promoter/reporter constructs showed 10-fold higher activity than control in REF-1 (rat), A31 (mouse) and CHO (hamster) cells. The region contains a TATA sequence and multiple potential transcription factor binding sites. Promoter activity was dose-dependently inhibited by cotransfection with either ras or mos oncogenes, but oncogene inhibition was reversed and the overall activity increased when cells were treated with the MAP kinase kinase (MKK) inhibitor PD98059. An NF-1 and Yi-like site, identified in the minimal promoter region, showed different mobility shift patterns when normal and transformed cell nuclear extracts are compared. Mutation of the NF-1 site reduced Cktsf1b1 promoter activity 25%, while mutation of the Yi-like site destroyed all the activity. Our results indicate that the expression of Cktsf1b1, a gene associated with early development and cell transformation, is sensitive to MKK levels and may be regulated via multiple transcription factor complexes.

DRM/GREMLIN (CKTSF1B1) maps to human chromosome 15 and is highly expressed in adult and fetal brain.

We have mapped and characterized the human homolog of Drm/Gremlin (CKTFS1B1), a member of a family of BMP antagonists that have been linked to both developmental and transformation-related functions. By screening a human cDNA library, we isolated a 3.3-kb cDNA containing the 552-bp region encoding the human DRM protein. CKTFS1B1 was localized on human chromosome 15q13--> q15 by somatic cell hybrid analysis and, more precisely, using radiation hybrids, to a region of markers linked to SGNE1, secretory granule neuroendocrine protein 1 and RYR3, the ryanodyne receptor 3. Northern blot analysis showed the presence of a single DRM-specific mRNA expressed in different human tissues, including brain, ovary, intestine and colon. In the brain, DRM expression is associated with the region localized around the internal capsule in the large subcortical nuclei. DRM appears to be predominantly expressed in normal cells and tissues, including normal neurons, astrocytes and fibroblasts. Interestingly, we detected DRM expression in normal cells obtained from several patients, but not in tumor cell lines established from the same patients. The data suggest that down-regulation of DRM is associated with tumor progression, and support the hypothesis that human DRM may play an important role during both neuroembryological development and carcinogenesis.

Biosynthesis, post-translation modification, and functional characterization of Drm/Gremlin.

Down-regulated by mos (Drm)/Gremlin is a highly conserved protein whose properties and expression pattern suggest a role in early development, tissue-specific differentiation, and cell transformation. We have investigated the biosynthesis and processing of Drm expressed endogenously in rat fibroblasts or overexpressed following transient or stable transfection. Analysis of metabolically labeled cells revealed that Drm exists in secreted and cell-associated forms that exhibit similar mobilities in SDS-polyacrylamide gel electrophoresis. Protein analysis indicated that Drm is present in two major species: a slow migrating glycosylated form and a nonglycosylated form. Both forms of Drm are able to undergo phosphorylation. Drm is released into the media within 30 min of synthesis and is detectable for up to 4-5 h, whereas the cell-associated form has a half-life of about 1 h. Confocal immunofluorescent microscopy indicates that Drm is present both on the external surface of expressing cells, as well as within the endoplasmic reticulum and the Golgi. Both glycosylated and nonglycosylated forms of Drm exhibit identical distributions and are able to antagonize bone morphogenetic protein signaling. Like the soluble form, the cell-associated forms are capable of binding (125)I-bone morphogenetic protein-4. These properties are consistent with a role for Drm in interfering with signaling and indicate that Drm may act at the cell surface during tissue development and transformation.

[Cathepsins L and B and their endogenous inhibitors in embryonal fibroblasts transformed by different genes]. / Katepsiny B i L i ikh éndogennye ingibitory v émbrional'nykh fibroblastach, transformirovannykh razlichnymi genami.

Expression of cysteine proteinases, cathepsins L and B, and their inhibitors was studied out in three model systems of rat embryo fibroblasts, sequentially immortalized and transformed by different genes. In Model I rat embryo fibroblasts were immortalized with DNA of early region of simian adenovirus SA7 (clone REF-1) and then transformed by c-Ha-ras oncogene (REF-2EJ; malignant transformation). In Model II and III, the immortalized fibroblasts (clone IE5) were obtained by transfection with the polyoma virus LT gene and the clone IE5 used lost this gene; the malignant transformation was achieved by transfection with the E7 gene (clone trF8; Model II) and E6/E7 genes ¿clone A5E5(pC7-1); Model III]¿ of human papilloma virus types 16 and 18 respectively. In Model I, the increase in the total cathepsin L and B activity was correlated with the stages of transformation, at the same time, in Models II and III, this activity in immortalized IE5 fibroblasts was higher than at transformation stage. The activity of cathepsin L in lysates of transformed fibroblasts--REF-2EJ, significantly exceeded this activity both in transformed cells trF8 and A5E5(pC7-1)(6- and 10-fold, respectively). In cell cultures of Models I and II, the increases in secreted activity of cathepsins L and B were correlated with the stages of fibroblasts transformation, but in cultures of Model III, this activity at the stage of malignant transformation was lower than that the stage of immortalization. Therefore, the activities of cathepsins L and B were expressed to varying degrees at different stages of oncogenic transformation and the expression of their activities were dependent on type of transforming gene. It was established that changes in proteolytic potential were correlated with differences in the transforming phenotype of cell clones. An endogenous inhibitor(s) of cysteine proteinases was found in conditioned media of all type cell cultures. Expression and inhibitory properties of this inhibitor(s) were different at distinct stages of transformation.

Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

Plasminogen activators, kallikrein-like proteinase and type I and type IV collagenases at various stages of oncogenic transformation.

Comparative studies of membrane-associated, intracellular and secreted activities of serine (uPA, kallikrein-like proteinase) and metalloproteinases (type I and IV collagenases) were carried out on rat embryo fibroblasts, sequentially immortalized and transformed by two different genes. Using this experimental model it was shown that (1) activity of uPA was expressed at the stage of immortalization solely; (2) intracellular and secreted activity of type I and IV collagenases decreased during process of transformation, (3) kallikrein-like proteinase activity was not revealed either in the primary or in transformed cells, (4) Z-Phe-Arg-MCA hydrolysis was the result of the action of cysteine proteinases alone; the increase in this activity was correlated with the stages of oncogenic transformation of fibroblasts.

Activation of the mitogen-activated protein kinase cascade in response to the temperature inducible expression of v-mos kinase.

We have characterized activation of the MAP kinase cascade in an inducible system in response to the temperature-sensitive (ts) expression of the v-mos oncogene. Transformation of immortalized rat embryo fibroblasts by a ts isolate of Moloney murine sarcoma virus (Mo-MuSVts110) constitutively activates MAP kinases (ERK-1 and ERK-2) and MAP kinase kinases (MKK-1 and MKK-2) only at the permissive temperature when v-mos kinase is present and active. Following a shift of the ts-transformed, serum-starved cells from the nonpermissive to permissive temperature, MAP kinases and both MKK-1 and MKK-2 are activated within 1-2 h, concurrent with the reappearance of active mos kinase. Raf-1 kinase activity increases more slowly in response to the reappearance of v-mos, and the mobility shift indicative of hyperphosphorylation was only detected 18 h after the temperature transition. Our data show that MAP kinase cascade activation is an early event following the reappearance of v-mos expression and v-mos kinase activity upon temperature shift, while the first manifestation of morphological transformation appears 24 h after the shift to permissive temperature. These results support the hypothesis that mos acts through the MKK to induce cell transformation.

Proteolytic enzymes at various stages of oncogenic transformation of rat fibroblasts. I. Aspartyl and cysteine proteinases.

Aspartyl and cysteine proteinases at distinct stages of carcinogenesis were analyzed in rat embryo fibroblasts, sequentially immortalized and transformed by 2 different genes: the early region of simian adenovirus SA7 and c-Ha-ras oncogene. The dynamics of expression and distribution of proteinases throughout the transformation process were examined. It was shown that in immortalized and transformed cells the activities of the aspartyl and cysteine proteinases were expressed to a variable degree and that the expression was dependent on cell-propagation time in vitro. The increase in activity both of cathepsin-D-like aspartyl proteinase and of cathepsin-L- and -B-like cysteine proteinases in cell lysates was correlated with the stages of fibroblast transformation (immortalization and tumorigenic transformation). In all cell types the major part of cysteine proteinases was localized inside the cell, while the cathepsin-D-like proteinase was apparently predominant among secreted proteinases. The cathepsin-L-like proteinase accounts for the major part of the cysteine-proteinase activity as measured by Z-Phe-Arg-MCA hydrolysis. We suggest that considerable portions of the cathepsin-D- and -L-like proteinases in all cell lines studied are secreted as a complex with inhibitor(s) and that inhibitor expression plays an important role in regulating the activity of cathepsin-D-like proteinase at different stages of transformation. Cathepsin-L-like proteinase is probably secreted in the precursor form.

Transformation-resistant mos revertant is unable to activate MAP kinase kinase in response to v-mos or v-raf.

To study the mechanism by which v-mos induces cell transformation, we generated a transformed rat cell line (DTM) containing two functional copies of mos, one encoding the p37v-mos of the m1 wild-type strain of Moloney murine sarcoma virus (Mo-MuSV) and the other the p85gag-mos fusion protein of the ts110 mutant of Moloney murine sarcoma virus. Subsequently, we isolated a revertant cell line (F-1) following transfection of DTM with a mutant retroviral construct (pIC4Neo) carrying a selectable marker. Like DTM, the F-1 revertant contained two integrated copies of v-mos, expressed mos containing viral RNA, and contained rescuable transforming viruses. The revertant did not grow in soft agar, showed a greatly reduced ability to form tumors in nude mice, and exhibited organized tubulin and actin structures similar to those found in normal cells. Revertant cells were resistant to retransformation by v-mos and v-raf but could be retransformed by v-ras. MAP kinase (ERK-2) and MAP kinase kinase (MKK-1) activity, which are constitutively elevated in v-mos- and v-raf-transformed cells, exhibits levels in the F-1 revertant similar to those seen in nontransformed cells. F-1 and normal REF-1 cells express elevated levels of protein phosphatases in comparison to DTM cells. In vivo treatment with okadaic acid, a potent protein phosphatase inhibitor, leads to an increase in MKK-1 and MAP kinase activity in F-1 cells but not in REF-1. The results support the hypothesis that mos acts through the MAP kinase cascade (MKK-1 and ERK-2) to induce cell transformation and that blocking v-mos activation of that cascade (possibly because of increased levels of phosphatase) prevents transformation.

[Expression of cathepsins D and L during neoplastic transformation of fibroblasts]. / Issledovanie ékspressii katepsinov D i L v protsesse onkogennoi transformatsii fibroblastov.

Study of the expression of the aspartyl cathepsin D-like and cysteine cathepsin L-like proteinases was carried out on a model system of rat embryo fibroblasts. The model system employed makes it possible to distinguish two discrete successive stages of transformation in vitro: immortalization and tumorigenic transformation. The dynamics of expression and subcellular distribution of proteinases throughout the transformation process was followed. It was shown that in immortalized and transformed cells the activities of the aspartyl and cysteine proteinases were expressed to a variable degree and the expression was dependent on the time of cell cultivation. The increase in both the aspartyl cathepsin D-like proteinase and cysteine cathepsin L- and B-like proteinase activities was correlated with the stage of fibroblast transformation. At all stages studied of transformation, the major part of cathepsin L-like proteinase activity was localized within the cell, while among secreted proteinases the cathepsin D-like proteinase was apparently predominant. It was found that the secreted cathepsin D-like proteinase in all cell cultures studied was complexed with the inhibitor.

Modulation of pp60v-src and pp60c-src expression in Rous sarcoma virus-transformed hamster fibroblasts transfected with activated N-ras.

Three phenotypically different hamster cell lines transformed with Rous sarcoma virus (RSV) were transfected with plasmid DNA containing an activated N-ras oncogene, and nine clones expressing various levels of p21N-ras were characterized. We examined the effects of p21N-ras on expression and kinase activity of resident src proteins by using a variety of assays that allowed us to discriminate between viral and cellular src proteins. In eight clones with a 10- to 20-fold increase in p21N-ras levels relative to the endogenous protein, we observed a marked reduction in the synthesis and kinase activity of p60v-src. This decrease correlated with transcriptional downregulation of RSV genomic and v-src subgenomic mRNAs. In the same cells, we found a concomitant accumulation of p60c-src and, accordingly, an increase in its protein kinase activity without an apparent increase in c-src mRNA levels. Therefore, modulation of viral and cellular src proteins in cells overexpressing p21N-ras appeared to result from two distinct effects: a downregulation of long terminal repeat-driven transcription and a more complex interaction with cellular effectors that control the stability of p60c-src. Such modulation also seemed to depend on the levels of p21N-ras and, possibly, on host-cell factors, since it was not observed in the third cell line, in which the relative increase in p21N-ras was only 2.5-fold to fivefold.

C-ets-1 protooncogene expression alters the growth properties of immortalized rat fibroblasts.

Ets family genes have been cloned and characterized from a variety of species ranging from human to Drosophila. The ets proteins encode transcription factors that activate transcription via specific binding to GGAA core sequence present in various promoter/enhancers. To investigate the role of ets protooncogene expression on the growth properties of rat embryo fibroblasts (REF), we constructed and introduced ets expression vectors into primary, as well as immortalized REF cells. The transfected cells contained multiple copies of the vector DNA, and the Northern blot analysis demonstrated overexpression of the c-ets-1-specific mRNA. Although the expression of the ets genes was unable to immortalize primary rat embryo fibroblasts, the expression of ets-1 in REF-1 cells enabled their growth in serum-free medium and effected tumorigenic activity in nude mice.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. III. Dissociation of the properties in N-ras-transfected RSV-SR-transformed cells.

We have previously shown that RSV-SR-transformed hamster cells acquire high resistance to H2O2, i.e. the cytotoxic product of activated macrophages (H2O2R) and that they begin to secrete PGE (PGES), thus inactivating the CTA of NK cells. Among normal cells, the same phenotype is expressed in activated macrophages. In all our RSV-transformed cells these 2 properties were jointly expressed and correlated with high tumorigenicity and experimental metastasizing of these cells. We now show that transfection of 3 RSV-SR-transformed cell strains with activated N-ras leads either to complete inhibition of the H2O2R + PGES phenotype in all clones of one strain, or to inhibition of PGES only in the majority of clones of 2 other strains. Unexpectedly, the complete or partial inhibition of this phenotype did not alter the high tumorigenicity of 2 strains of these cells, but lower tumorigenicity was evident in almost all clones of the third strain (as well as in some gene-neo-transfected clones of these strains). The loss of PGES made these cells susceptible to the CTA of NK cells, while the loss of H2O2R did not alter their resistance to the CTA of macrophages. Expression of the H2O2R + PGES phenotype was retained in all cloned variants of control, gene-neo-transfected cells. The possible relation of the N-ras gene to regulation of src gene activities in RSV-SR-transformed cells is discussed.

Changes in surface relief of suspended cells are morphological signs of the initial stage of neoplastic transformation in fibroblastic monolayer cultures.

The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.

[Cysteine proteinases at various stages of neoplastic transformation of rat fibroblasts]. / Issledovanie tsisteinovykh proteinaz na raznykh étapakh onkogennoi transformatsii fibroblastov krys.

Cysteine-dependent proteinases were studied in a model system of rat embryonal fibroblasts which enabled to evaluate two discrete steps of malignant transformation in vitro: immortalization and tumoral transformation. Dynamics of intracellular and secreted activities of cysteine proteinases was estimated in relation to the stage of transformation and duration of cells cultivation. Activation of cysteine proteinases cathepsins L and B correlated with developed transformation of fibroblasts; each step of the process was characterized. Intracellular and secreted activities of the proteinases were distinctly increased in immortalized cells as compared with primary fibroblasts. The highest activity of the enzymes was detected both in intra- and extracellular fractions at the stage of complete fibroblast transformation. Cysteine-dependent cathepsin L-like proteinase was mainly responsible for intracellular and secreted activities of the fibroblast cultures studied. This proteinase was found to be produced in the form of precursor in all the cell strains studied and/or main part of the enzyme developed complexes with an inhibitor.

[A new recipient cell line for transfection of biologically active oncogenes]. / Novaia retsipientnaia kletochnaia liniia dlia transfektsii biologicheski aktivnykh onkogenov.

The properties of the new immortalized rat cell line (REF-1) were analyzed. These cells can be used as recipient ones in transfection assays. REF-1 cells never convert spontaneously to transformed phenotype during long-term passages in vitro unlike NIH3T3 cells. This peculiarity allowed to use REF-1 cells for identification of oncogenes, which induce slow-growing tumors. The following oncogenes were used in gene transfection experiments in order to test their effects: activated human EJ-ras; N-ras; v-myc; v-mos; activated tpr-met and c-hu-met. REF-1 cells, transfected with the members of ras family; v-mos and tpr-met were found to be transformed in vitro and induce tumors in nude mice, on the contrary of c-hu-met- and v-myc-transfected cells, which are non-tumorogenic. A number of clonal cell lines carrying different oncogenes were obtained. The detailed analyses of integration and expression of exogenous sequences of different oncogenes has been presented in 18 clonal cell lines.

[Activity of topoisomerase I and endonucleases in cells transfected by a ras oncogene]. / Aktivnost' topoizomerazy I i éndonukleaz v kletkakh, transfitsirovannykh onkogenom ras.

Activities of nuclear endonucleases and topoisomerase I were measured in rat fibroblasts which were at the stages of tumor transformation: control embryonal fibroblasts--CEF; cells immortalised by transfection of S1A segment of SA7 adenovirus--REF-1; intermedius cells transfected once by EJras oncogene--REF-1EJ; and cells transformed after the second transfection by the same oncogene--REF-2EJ. The topoisomerase I and Ca2+, Mg2+-dependent endonuclease was most decreased at the stage of immortalised cells, and the intermedius stage (REF-1EJ) was characterized by the lower activity of Ca2+, Mg2+-dependent endonuclease. The highest activity of Mn2+-dependent endonuclease is seen in REF-2EJ cells. In model experiments the ability of Ca2+, Mg2+-dependent endonuclease to split non-stochastically the EJras oncogene inserted into pBR322 plasmid was shown. The role of the investigated enzymes in the restriction of plasmid integration, cellular immortalisation and recombination of plasmids with chromosomes during cell transformation is discussed.

[Model systems of carcinogenesis in vitro: the interaction of the ras oncogene with immortalized cells]. / Model'nye sistemy kantserogeneza in vitro: vzaimodeistvie onkogena ras s immortalizovannymi kletkami.

Gene transfer experiments have shown that ras effector functions are sufficient to transform cells from a variety of established lines (e. g., mouse NIH3T3 cells). In contrast, primary cells and early passage rodent cells can be transformed by ras oncogenes only at low frequencies, unless cotransfected with collaborating genes such as adenovirus early region IA (EIA) or myc retroviral oncogene homologue. Primary rat embryo fibroblasts (REF) were chosen as a model for the analysis of multistep cellular transformation. Transfection of REF, immortalized by early region of simian adenovirus SA7 with c-Ha-ras oncogene cannot induce their morphological transformation. This phenomenon is observed only after second transfection with the same oncogene. These different cell lines can be used for further analysis of the mechanisms of carcinogenesis.

[Transfection of a plasmid with a retrovirus promoter: distribution of sequences in the genome of induced tumors]. / Transfektsiia plazmidy s retrovirusnym promotorom: raspredelenie posledovatel'nostei v genome indutsirovannykh opukholei.

The structure of the integration site of plasmid with LTR of Rous sarcoma virus (pLTR1,5) in the genome of nude mice tumors, induced as a result of N1H3T3 cells' implantation, cotransfected by pLTR1,5 with the DNA of malignant human glioma cells, carrying amplified c-Ha-ras genome, has been studied. The restriction map of the investigated region of the cell genome was obtained. Molecular cloning of the integrated plasmid and adjacent cell sequences has been carried out. It was shown that the exogenic vector in the DNA of the tumor cells is included in BamHI structure of the repeat of mice genome.