Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Lysosomal cathepsin B: correlation with metastatic potential.

Although lysosomal enzymes are implicated in the processes of tumor invasion and metastasis, their cellular origin within the tumor is unclear. The activity of the lysosomal proteinase cathepsin B is significantly elevated in a variant of the B16 melanoma with high metastatic potential. The cathepsin B activity is localized to the lysosomes of the tumor cells.

Tumor cell-platelet aggregation: induced by cathepsin B-like proteinase and inhibited by prostacyclin.

The ability of tumor cells to metastasize may be related to their ability to promote aggregation of host platelets. The use of inhibitors of cysteine proteinases resulted in parallel inhibition of B16 amelanotic melanoma-induced platelet aggregation and of a cathepsin B activity. The antimetastatic agent prostacyclin inhibited platelet aggregation induced by the tumor cells and by papain, a cathepsin B-mimicking agent.

Inhibition by prostacyclin of the tumor cell-induced platelet release reaction and platelet aggregation.

Prostacyclin was examined for its inhibitory effects on the tumor cell-induced platelet release reaction. Prostacyclin inhibited in a dose-dependent manner tumor cell-induced release of platelet dense granules and alpha-granules concomitant with an inhibition of platelet aggregation. Release was determined by assay of biochemical markers (serotonin for dense granules and beta-thromboglobulin for alpha-granules). A tenfold higher concentration of prostacyclin was required to inhibit completely serotonin release as compared to the concentration required for beta-thromboglobulin release. Correlative ultrastructural studies demonstrated that prostacyclin at doses of over 10 ng/ml inhibited the ultrastructural changes associated with tumor cell-induced platelet shape change and platelet granule release. Platelet aggregates exhibited the retention of granule reservoirs that could potentially be involved in long-term release of biologically active substances.

Platelet enhancement of tumor cell adhesion to subendothelial matrix: role of platelet cytoskeleton and platelet membrane.

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.

Pericellular pH affects distribution and secretion of cathepsin B in malignant cells.

Redistribution of lysosomes to the cell surface and secretion of lysosomal proteases appear to be general phenomena in cells that participate in local proteolysis. In the present study, we have determined whether malignant progression affects the intracellular distribution and secretion of the lysosomal protease cathepsin B in three model systems, each of which consists of cell pairs that differ in their degree of malignancy. The intracellular distribution of vesicles staining for cathepsin B was evaluated by immunofluorescent microscopy and the secretion of cathepsin B was evaluated by two complementary techniques: stopped assays of activity secreted into culture media; and continuous assays of activity secreted from viable (> or = 95%) cells growing on coverslips. We observed that the intracellular distribution of cathepsin B+ vesicles was more peripheral in the cells of higher malignancy in all three model systems and that active cathepsin B was secreted constitutively from these cells. Because an acidic pericellular pH has been shown to induce translocation of lysosomes in macrophages and fibroblasts, we evaluated the intracellular distribution of cathepsin B+ vesicles and secretion of cathepsin B in cell pairs incubated at slightly acidic pH. Acidic pericellular pH induced a redistribution of cathepsin B+ vesicles toward the cell periphery. In the more malignant cells, this resulted with time in reduced intracellular staining for cathepsin B and enhanced secretion of active cathepsin B. Translocation and secretion of cathepsin B were dependent on a functional microtubular system. Both the redistribution of cathepsin B+ vesicles toward the cell surface induced by acidic pH and the constitutive and acidic pH-induced secretion of active cathepsin B could be inhibited by microtubule poisons and stabilizers. We suggest that the redistribution of active cathepsin B to the surface of malignant cells and its secretion may facilitate invasion of these cells.

Cathepsin B-like activity in viable tumor cells isolated from rodent tumors.

The cathepsin B-like cysteine proteinase activity which has been implicated in tumor malignancy has been attributed to several cellular sources, including viable tumor cells, necrotic tumor cells, and host-inflammatory cells. We have isolated subpopulations of cells from eight rodent tumors of five histological types, using centrifugal elutriation, and verified the cellular composition of the subpopulations cytologically. Ninety-two % or greater of the cathepsin B-like activity was associated with the isolated fractions containing greater than or equal to 95% tumor cells of 86 +/- 2% (SE) viability (beta fractions). The isolated fractions consisting of necrotic tumor cells and inflammatory cells (alpha fraction) apparently contain a cysteine proteinase inhibitor, since both cathepsin B-like and cathepsin H activities in the beta fraction of B16 amelanotic melanomas could be inhibited by addition of the alpha fraction.

A new in vitro model for investigation of tumor cell-platelet-endothelial cell interactions and concomitant eicosanoid biosynthesis.

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A2 balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostaglandin F1 alpha concentration; an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A2 production by platelets; indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: little or no platelet or tumor cell adhesion to gelatin beads alone, a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.

Cathepsin B activity in B16 melanoma cells: a possible marker for metastatic potential.

In solid s.c. tumors of a variant of the murine B16 melanoma with high metastatic potential (B16F10), there was a 2- to 7-fold elevation of lysosomal cathepsin B activity when compared to the B16F1 variant with low metastatic potential. The highest activities (based on either protein or DNA) of cathepsin B were found in tumors of less than 1 g. When B16F1 and B16F10 melanoma variants were grown in tissue culture, the metastatic differential in cathepsin B activity was lost as the cells were subcultured. However, this differential in cathepsin B activity could be restored by reestablishing the cultured cells as s.c. tumors. The activities of four other lysosomal enzymes (cathepsin D, beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase) showed little evidence of a positive correlation with the metastatic potential of the B16 melanoma variants. Eighty to 90% of cathepsin B activity has been localized to a fraction containing viable tumor cells which was isolated by centrifugal elutriation. In contrast, only 50% of cathepsin D activity was in the viable tumor cell fraction, and from 30 to 70% of beta-N-acetylglucosaminidase, beta-glucuronidase, and acid phosphatase. Elevated levels of cathepsin B in the high metastatic B16F10 variant are consistent with the idea that cathepsin B may play a direct or a regulatory role in tumor metastasis.

Cathepsin B expression and localization in glioma progression and invasion.

The poor prognosis of human malignant gliomas is due to their invasion and recurrence, the molecular mechanisms of which remain poorly characterized. We have accumulated substantial evidence implicating the cysteine protease cathepsin B in human glioma malignancy. Increases in cathepsin B expression were observed throughout progression. In primary brain tumor tissue, transcript abundance (Northern blot analysis) increased in low-grade astrocytoma to high-grade glioblastoma from 3- to 6-fold, respectively, above normal brain levels. This increase correlated with increases in protein abundance (from + to ) as measured by immunohistochemistry. Furthermore, in glioblastoma cell lines increases in transcript abundance (ranging from 3- to 12-fold) were accompanied by increases in enzyme activity (44-133 nmol/min x mg protein). Altered subcellular localization was observed both immunohistochemically and by indirect immunofluorescence confocal microscopy and was found to correlate with increased grade. In addition, this increase in cathepsin B expression and altered subcellular localization correlated with histomorphological invasion and clinical evidence of invasion as detected by magnetic resonance imaging. These data support the hypothesis that cathepsin B plays a role in human glioma progression and invasion.

Properties of a plasma membrane-associated cathepsin B-like cysteine proteinase in metastatic B16 melanoma variants.

Activities of a cathepsin B-like cysteine proteinase have previously been observed to correlate with the malignancy of several animal and human tumors. Plasma membrane fractions of some of these tumors have been found to be enriched in cathepsin B-like activity. We have determined the subcellular distribution of this enzyme and three additional lysosomal hydrolases (cathepsin H, beta-hexosaminidase, and beta-glucuronidase) in normal murine liver and six metastatic variants of the B16 melanoma. The tissues were fractionated initially by differential centrifugation followed by Percoll density gradient centrifugation of the light mitochondrial fraction. Two fractions were obtained: an L-2 fraction enriched in all four lysosomal hydrolases; and an L-1 fraction enriched in a marker enzyme for the plasma membrane. Cathepsin B-like and beta-hexosaminidase activities, but not the other hydrolase activities, were also found to be enriched in the L-1 fractions of the metastatic B16 tumors. We explored the nature of the association of the cathepsin B-like activity with the plasma membrane using fractions from the spontaneously metastatic B16 amelanotic melanoma. Activity could not be dissociated from the plasma membrane fraction by washing with a physiological salt solution suggesting that it was not adsorbed to this fraction nonspecifically, nor could it be displaced by mannose 6-phosphate or other sugars which compete for binding to the known lysosomal receptors. High salt concentrations, low concentrations of the mild detergent saponin, mild acidification, or phosphatidylinositol-specific phospholipase C did not elute the cathepsin B-like activity. However, activity was eluted by exposure to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a detergent used in the purification of integral membrane proteins. The B16 amelanotic melanoma plasma membrane-associated cathepsin B-like activity had a slightly higher pH optimum and was resistant to inactivation by neutral pH and to inhibition by three low molecular weight inhibitors of cysteine proteinases. The Ki values for inhibition by leupeptin and stefin A were 20-fold higher. The presence of a cathepsin B-like cysteine proteinase at the surface of metastatic tumor cells, particularly in a form which can retain activity at physiological pH and retain activity in the presence of extracellular proteinase inhibitors, may contribute to the focal dissolution of the extracellular matrix observed at sites of contact with invading tumor cells.

Cathepsin B to cysteine proteinase inhibitor balance in metastatic cell subpopulations isolated from murine tumors.

Our laboratories have previously demonstrated that the malignancy of human and animal tumors is associated with increases in cathepsin B activity, due in part to increases in cathepsin B-specific RNA transcripts and in part to decreased regulation by the endogenous low molecular weight cysteine proteinase inhibitors (CPIs). In this study we have extended these observations to tumor cell subpopulations of B16 amelanotic melanoma (B16a) and Lewis lung carcinoma (3LL) isolated by centrifugal elutriation. B16a subpopulations exhibited a 10-fold differential in lung colonization potential, whereas 3LL subpopulations exhibited no differential. In the B16a subpopulations, cathepsin B activities, total cellular and plasma membrane-associated, corresponded positively (4- and 10-fold increase, respectively) with their lung colonization potentials. CPI activities, total cellular and plasma membrane-associated, corresponded inversely (2- and 5-fold decrease, respectively) with the lung colonization potential of the B16a subpopulations. In the 3LL subpopulations, neither cathepsin B nor CPI activities changed. In the plasma membrane fractions of all 3LL subpopulations the ratio of cathepsin B activity to CPI activity was less than 1, whereas in the plasma membrane fractions of all B16a subpopulations the ratio was 1 or greater. In the plasma membrane fractions of the B16a subpopulations of higher lung colonization potential the ratios were 2.5 and 7, indicating that the levels of endogenous CPIs in these fractions may not be sufficient to regulate cathepsin B activity. Cathepsin B mRNA levels were not increased in the B16a subpopulations expressing increased cathepsin B activity. Thus increased cathepsin B activity in these subpopulations was apparently due not to increased synthesis but to decreased regulation by the endogenous CPIs. These results suggest that membrane-associated cathepsin B and CPIs may both play a role in the expression of the experimental metastatic phenotype.

Role of platelet membrane in enhancement of tumor cell adhesion to endothelial cell extracellular matrix.

Tumor cell adhesion to subendothelial matrix in the presence of platelets and plasma has been examined in vitro using an entirely homologous system of rat Walker 256 carcinosarcoma cells, matrix laid down by rat aortic endothelial cells and rat platelets and plasma. In the presence of platelets or platelets plus plasma, tumor cell adhesion was significantly enhanced when compared to adhesion in the absence of platelets. In the presence of plasma alone (0.1%), we observed no significant increase in tumor cell adhesion. In order to determine which platelet factors contribute to the enhancement of tumor cell adhesion by platelets, we subjected washed rat platelets to mechanical lysis or thrombin stimulation followed by centrifugation. The membrane fractions and supernatant fractions containing platelet attachment proteins were compared for their abilities to support tumor cell adhesion to subendothelial matrix. Platelet membranes were also recombined with platelet supernatant fractions to determine if platelet attachment proteins or platelet membranes required the presence of the other to enhance tumor cell adhesion. Platelet supernatant fractions which contained release reaction proteins (confirmed by polyacrylamide gel electrophoresis) did not enhance tumor cell adhesion. Purified thrombospondin, fibronectin, beta-thromboglobulin, platelet derived growth factor, and serotonin had no effect on tumor cell adhesion. Platelet membrane containing fractions affected tumor cell adhesion to subendothelial matrix as follows: (a) platelets formed an adhesive bridge between tumor cells and the subendothelial matrix as demonstrated by scanning electron microscopy; (b) intact platelets and thrombin stimulated platelets were the most effective at facilitating tumor cell adhesion; (c) preparations containing partially lysed platelet ghosts were more effective in supporting tumor cell adhesion to subendothelial matrix than were preparations containing completely lysed platelet membrane fragments; (d) recombination of platelet supernatant fractions with mechanically lysed platelets did not enhance their ability to support adhesion; (e) fixed platelets, either alone or in combination with platelet supernatant fractions, failed to enhance adhesion. These data indicate that platelet enhanced tumor cell adhesion appears to be dependent on platelet membrane factors including receptor mobility, rather than intraplatelet components.

Cell surface complex of cathepsin B/annexin II tetramer in malignant progression.

The cysteine protease cathepsin B is upregulated in a variety of tumors, particularly at the invasive edges. Cathepsin B can degrade extracellular matrix proteins, such as collagen IV and laminin, and can activate the precursor form of urokinase plasminogen activator (uPA), perhaps thereby initiating an extracellular proteolytic cascade. Recently, we demonstrated that procathepsin B interacts with the annexin II heterotetramer (AIIt) on the surface of tumor cells. AIIt had previously been shown to interact with the serine proteases: plasminogen/plasmin and tissue-type plasminogen activator (tPA). The AIIt binding site for cathepsin B differs from that for either plasminogen/plasmin or tPA. AIIt also interacts with extracellular matrix proteins, e.g., collagen I and tenascin-C, forming a structural link between the tumor cell surface and the extracellular matrix. Interestingly, cathepsin B, plasminogen/plasmin, t-PA and tenascin-C have all been linked to tumor development. We speculate that colocalization through AIIt of proteases and their substrates on the tumor cell surface may facilitate: (1) activation of precursor forms of proteases and initiation of proteolytic cascades; and (2) selective degradation of extracellular matrix proteins. The recruitment of proteases to specific regions on the cell surface, regions where potential substrates are also bound, could well function as a 'proteolytic center' to enhance tumor cell detachment, invasion and motility.

Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma.

Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). Inhibitors from human sarcoma were compared to those from human liver, a normal tissue in which the inhibitors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain and cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or isoform of cystatins. We purified two members of the cystatin superfamily (stefin A and stefin B) to homogeneity and determined their individual inhibitory properties. Stefins B from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepsins B, H and L and human and murine tumor cathepsin B. The Ki for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Cancer is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.

Differentiating agents regulate cathepsin B gene expression in HL-60 cells.

We utilized HL-60 cells as a model system to examine the regulation of ctsb gene expression by differentiating agents. Inducers of monocytic differentiation [phorbol ester (PMA), calcitriol (D3), and sodium butyrate (NaB)] and inducers of granulocytic differentiation [all-trans retinoic acid (RA) and 9-cis retinoic acid (9-cis RA)] increase ctsb mRNA levels in a dose-dependent manner as determined by Northern blot hybridization. D3 and retinoids exert additive effects, suggesting that these agents act in part through distinct pathways. Actinomycin D decay experiments indicate that D3, NaB, RA, and 9-cis RA do not alter mRNA stability. In contrast, PMA markedly increases the half-life of ctsb mRNA. In transient transfection assays, PMA and NaB both stimulate transcription of the luciferase reporter gene placed under the control of ctsb promoter fragments. Thus, inducers of HL-60 cell differentiation can regulate the expression of the ctsb gene at both transcriptional and posttranscriptional levels.

Imaging proteolysis by living human breast cancer cells.

Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through the use of quenched-fluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin B-selective cysteine protease inhibitor, intracellular fluorescence was decreased approximately 90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence approximately 50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1) a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2) the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.

Identification of two new exons and multiple transcription start points in the 5'-untranslated region of the human cathepsin-B-encoding gene.

Transcripts for cysteine protease cathepsin B (CTSB) were found to be highly variable in the 5'-UTR (untranslated region). In cDNA clones from a human gastric adenocarcinoma cDNA library, we have identified two new exons (designated 2a and 2b) between exons 2 and 3 in the 5'-UTR of the gene. All of the exons of the 5'-UTR could be alternatively spliced to produce several transcript species. In addition, transcription was initiated from more than one promoter region. Using RT-PCR (reverse transcription-polymerase chain reaction) and primer extension assays, CTSB mRNA species were found to differ among tissues and between a glioblastoma sample and a cell line derived from it. Exons 2a and 2b were detected more frequently in tumor samples than in matched normal samples. Thus, factors related to the cell differentiation and environment seem likely to determine the types of transcripts that are expressed which in turn could influence the overall steady-state level of CTSB mRNAs and their rate of translation. Interestingly, at least three upstream translation initiation codons were observed and could constitute a means of controlling translation initiation.

Human gastric adenocarcinoma cathepsin B: isolation and sequencing of full-length cDNAs and polymorphisms of the gene.

Four full-length cDNA clones coding for preprocathepsin B were isolated from a human gastric adenocarcinoma cDNA library (AGS 1-6-30-1) and analyzed for possible sequence modifications that might be linked to altered intracellular trafficking and secretion of cathepsin B (CTSB) in malignant tumors. Comparison of AGS 1-6-30-1 cDNAs with human kidney/hepatoma cDNAs revealed: (1) three potential N-glycosylation sites instead of two, (2) a nucleotide (nt) substitution in the coding region for the propeptide from GTG to CTG which would result in a Val26-->Leu change, (3) three silent nt replacements in the coding region for the mature protein, (4) five single-nt differences in the 5'- and 3'-UTR (untranslated regions), (5) heterogeneity in the 5'-UTR, and (6) a 10-bp insertion in the 3'-UTR. The 10-bp insertion in the 3'-UTR may alter the stability of CTSB mRNA transcripts and thereby the expression of CTSB. These clones should be useful for expressing human tumor CTSB and analyzing the function of this enzyme in malignant progression. Two restriction-fragment length polymorphisms (RFLPs), EcoRI and TaqI, were detected by Southern blot analysis of genomic DNA from 36 unrelated Caucasians. Inheritance and distribution of the EcoRI alleles (13.0 and 11.0 kb) and the TaqI alleles (5.7 and 4.6 kb) indicated they were independent polymorphisms. In contrast to the EcoRI alleles of 13.0 and 11.0 kb observed in the population survey, genomic DNA from two AGS gastric adenocarcinoma subclones revealed two EcoRI alleles of 13.0 and 7.8 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

Exon 2 of human cathepsin B derives from an Alu element.

Transcripts for the cysteine protease cathepsin B are alternatively spliced in the untranslated regions (UTRs). We show that a cathepsin B probe containing 5'-UTR sequences hybridized to an RNA of approximately 300 nt in addition to the typical 2.2 and 4.0 kbp mRNAs. Within this 5'-UTR, exon 2 was found to be homologous to Alu repetitive elements. Specifically, exon 2 was part of an Alu element interspersed with the cathepsin B gene. The approximately 300 nt band that hybridized to our cathepsin B probe likely corresponds to Alu transcripts, which are known to accumulate in human cells. Indeed, a similarly migrating band was detected with an authentic Alu probe. Thus, we suggest that primary transcripts for cathepsin B contain Alu sequences which are preserved as exon 2 in some fully spliced mRNAs.