Expression and regulation of three lysosomal cysteine protease activities during growth of a differentiating L6 rat myoblast cell line and its nonfusing variant.

The expression of three lysosomal cysteine protease activities, cathepsins B, H, and L, was examined during differentiation of L6 rat myoblasts. Analyses of intracellular levels of these proteases in unfractionated homogenates prepared from cells at different stages of growth and in parallel HPLC-fractionated samples demonstrated a fusion-related increase in all three cathepsins. Analyses of total levels of endogenous inhibitor activity against purified cathepsin B demonstrated a threefold increase in the ratio of protease to inhibitor during myoblast-myotube formation; however, levels of inhibitor activity remained constant. Extracellular levels of cathepsin B, H, and L activities were also examined in the serum-free defined media of differentiating L6 cells. These studies demonstrated a fusion-related increase in extracellular levels of acid/pepsin-activated (i.e., latent) cathepsin L. While increases in intracellular and extracellular levels of cathepsin activities were temporally related to the fusion process, fusion may not be a prerequisite for increased expression, since the nonfusing L6 variant L6-D3 demonstrated high levels of intracellular cathepsins B and L and extracellular latent cathepsin L activities throughout growth. Taken together, these results support the hypotheses that fusion or fusion-related processes play an important role in the controlled expression of cathepsins in L6 myoblasts and that cathepsins, in turn, play an important role in myoblast-myotube differentiation.

Characterization of cysteine proteases and their endogenous inhibitors in MCF-7 and adriamycin-resistant MCF-7 human breast cancer cells.

The expression of three lysosomal cysteine proteases was examined in a lowly metastatic, MCF-7 human breast cancer cell line and its highly metastatic, Adriamycin-resistant variant, MCF-7/AdrR. While levels of cathepsin H activity were similar in all cell lines at each stage of growth, intracellular cathepsin B and L activities were highest in MCF-7/AdrR. These high levels were accompanied by growth-related increases in acid/pepsin-activatable cathepsin activity in the culture medium. Analyses of endogenous cathepsin B inhibitor activity in control and heat-treated cell homogenates after fractionation by fast protein liquid chromatography suggested that alterations in cystatin-like, cysteine protease inhibitor activities contribute to increased levels of cathepsin activities in metastatic MCF-7/AdrR cells.

Age-related changes and tissue distribution of parvalbumin in normal and dystrophic mice of strain 129 ReJ.

In murine muscular dystrophy, hindlimb muscle contains a functionally defective thiol protease inhibitor (TPI) which has been implicated in the onset and progression of the disease in mice. More recently, this protease inhibitor has been identified as parvalbumin, a calcium binding protein. In this study, a polyclonal antibody against mouse muscle parvalbumin was used to study the concentration and distribution of this protein in normal and dystrophic male mice at various ages. Immunodetection assays were used to screen extracts of hindlimb, forelimb, brain, heart, lung, liver, and kidney in 60-day-old normal and dystrophic male mice for parvalbumin content. Parvalbumin was detected in relatively high amounts in both hindlimb and forelimb muscle extracts, while much lower concentrations were detected in brains of normal and dystrophic animals. No parvalbumin was detected in the lung, liver, heart, or kidney extracts using the immunoassay. With aging, the parvalbumin concentration in hindlimb muscle of normal mice remained fairly constant for 90 days, whereupon the level increased at 120 days. In contrast, the parvalbumin concentration in hindlimb muscle of dystrophic mice decreased steadily with age to about 22%% of normal animals at 120 days. The parvalbumin content was also reduced in dystrophic brain.

Suppression of aryl hydrocarbon hydroxylase activity in human primary lung carcinoma x mouse hepatoma somatic cell hybrids.

Variants of the mouse hepatoma cell clone inducible for aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) (EC 1. 14. 14.1) activity and deficient in hypoxanthine guanine phosphoribosyl-transferase (EC 2.4.2.8), and human primary lung carcinoma cell clone noninducible for AHH activity and deficient in thymidine kinase (EC 2.7.1.21) were isolated. The variant lines characterized for AHH inducibility and drug resistant phenotype were utilized to study somatic cell hybrids for the expression of AHH induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In two hybrids AHH activity was not expressed. In view of these results we conclude that aryl hydrocarbon hydroxylase activity is suppressed in AHH noninducible human lung carcinoma x AHH inducible mouse hepatoma cell hybrids.

Induction of aryl hydrocarbon hydroxylase and demonstration of a specific nuclear receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in two human hepatoma cell lines.

Two established human hepatoma cell lines, Hep3B and HepG2, were examined for aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) induction and for the presence of the murine-equivalent aromatic hydrocarbon (Ah) receptor. Both cell lines demonstrated polycyclic aromatic hydrocarbon (PAH)-induced AHH activity; however, assay conditions for induction were different than those established for the control mouse hepatoma cell line, Hepa c1-9. When cytosols from either cell line were exposed to tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin [( 3H]TCDD) and analyzed on sucrose gradients with or without prior charcoal treatment, two peaks were observed at positions corresponding to 4-5 S and 8-9 S. The 8-9 S peak was identified as the probable human Ah receptor equivalent since, like the mouse Ah receptor, this peak: (a) was eliminated only by cytochrome P1-450 inducers; (b) was sensitive to protease digestion; and (c) was thermolabile. Levels of TCDD specifically bound in the 8-9 S peak for HepG2 and Hep3B were 27 and 34 fmol/mg cytosolic protein respectively. The level of TCDD specifically bound was not affected by charcoal treatment or by the addition of sodium molybdate, which is known to stabilize ligand binding to steroid receptors. Incubation of Hep3B or HepG2 cells with [H]TCDD at 37 degrees for 1 hr effected a redistribution of binding from the cytosol 8-9 S peak to a nuclear 6 S peak. The nuclear peaks from both human cell lines demonstrated similar sedimentation properties, temperature-dependence and inducer-specificity, as for the mouse nuclear Ah receptor. Appearance of nuclear 6 S binding is consistent with a temperature-dependent translocation process, supporting the observation that these human hepatoma cell lines contain a binding component which is similar to the mouse Ah receptor in structure and function during AHH induction.

Relation between cytochrome P450IA1 expression and estrogen receptor content of human breast cancer cells.

Multidrug resistance (MDR) in an MCF-7 human breast cancer cell line (MCF7/Adr) is associated with decreased drug accumulation and overexpression of P-glycoprotein as well as alterations in the levels of specific drug-metabolizing enzymes, including decreased activity of the phase I drug-metabolizing enzyme aryl hydrocarbon hydroxylase (AHH) and increased expression of the anionic form of the phase II drug-metabolizing enzyme glutathione S-transferase. Since the development of MDR in this MCF-7 cell line is also associated with a loss of estrogen receptors (ER), we have examined the expression of cytochrome P450IA 1, the gene encoding AHH activity, in other breast cancer cell lines not selected for drug resistance but expressing various levels of ER. These studies show that a relationship exists between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AHH activity and the ER content in a series of breast cancer cell lines. In these cell lines expression of AHH activity is regulated, at least in part, at the level of P450IA 1 RNA. While TCDD-specific binding proteins (Ah receptors) were found in each of the breast cancer cell lines, there was no apparent relation between the level of nuclear TCDD-binding proteins and the level of TCDD-inducible P450IA 1 expression. Previous studies from our laboratory have described an inverse relationship between levels of the anionic form of glutathione S-transferase and ER in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells.

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p less than 0.005), two had approximately the same, and two had significantly higher (p less than 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 +/- 0.09 for clone 1 (Hs-1) and 0.94 +/- 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p less than 0.005), two had approximately the same, and one had significantly higher levels (p less than 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).

Evidence that 2,3,7,8-tetrachlorodibenzo-p-dioxin induces NADPH cytochrome c (P-450) reductase in rat hepatoma cells in culture.

The lack of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) (EC 1.14.14.1) induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a clone of rat hepatoma (HTC cl-1) cells is not caused by the lack of nuclear Ah receptor or by a deficiency in the activity of NADPH-cytochrome c (P-450) reductase. Treatment of HTC cl-1 cell line with TCDD for 18 h in culture resulted in a reproducible 500-600% increase in reductase activity without concomitant expression in AHH activity. These data suggests that TCDD induces cytochrome c reductase activity and that the lack of inducible AHH activity in rat hepatoma cells could reflect a defect in the structural gene (s) encoding for cytochrome P1-450, or an Ah receptor with a faulty DNA binding domain.

Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells.

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice.

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.

Evidence for a defective thiol protease inhibitor in skeletal muscle of mice with hereditary muscular dystrophy.

The thiol protease inhibitor (TPI-d) from hind-limb skeletal muscle of dystrophic 60-day-old male mice (strain 129/ReJ/dy) has been purified to apparent homogeneity and compared with the thiol protease inhibitor (TPI-n) from hind-limb skeletal muscle of normal 60-day-old male littermates. While both TPI-d and TPI-n displayed identical properties on sodium dodecyl sulfate-polyacrylamide gels (14,800 relative mass), analytical isoelectric focusing gels (pI 4.5), and high performance liquid chromatography columns, TPI-d was unable to inhibit papain and cathepsin B after purification by isoelectric focusing. However, a component in the purified TPI-d preparation with an isoelectric point of 4.9 initially masked the functional state of TPI-d, using papain when assayed with the test proteases papain and cathepsins H and L. This inhibitory component was absent from TPI-n preparations. Pure TPI-d was also unable to inhibit in vitro myosin hydrolysis by cathepsin B, whereas TPI-n completely blocked cathepsin B catalyzed myosin hydrolysis. Given the central role of the thiol proteases, especially cathepsin B, in intracellular protein metabolism and the possibility that uncontrolled thiol protease activity in muscle leads to muscle protein breakdown and dystrophy, our data suggest that a modified (defective) thiol protease inhibitor (TPI-d) may be (one of) the end product(s) of the dystrophy gene in mice with the hereditary form of the disease.

Analysis of Ah gene locus by somatic cell hybridization: expression of Ah regulatory gene product for 2,3,7,8,-tetrachlorodibenzo-p-dioxin in mouse L-cell x mouse hepatoma cell hybrids.

Properties of the aryl hydrocarbon hydroxylase (AHH) enzyme system were examined in polycyclic aromatic hydrocarbon (PAH) -noninducible L-cell x PAH-inducible hepatoma (Hepa) mouse cell hybrids. In hybrids, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces AHH activity. The levels of maximal TCDD-induced AHH activity in the hybrids and the Hepa parent are similar, although a greater concentration of TCDD is required for expression in the hybrids. This concentration difference appears to reflect dilution of AHH-associated gene products by the L-cell parent rather than altered gene expression. The regulatory gene product, the Ah receptor, is expressed similarly in the hybrids and Hepa parent. Both demonstrate specific, high-affinity binding of [3H]TCDD to an equivalent number of receptor sites per cell. These results suggest that the molecular mechanism of phenotypic resemblance to the inducible Hepa parent (i.e., "dominance") in the mouse L-cell x Hepa hybrids involves expression of only the Hepa Ah gene complex.

Defective binding of 3-methylcholanthrene to the Ah receptor within C3H/1OT1/2 clone 8 mouse fibroblasts in culture.

C3H/1OT1/2 clone 8 mouse fibroblasts (C3H/1OT1/2 cells) exhibit induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) when exposed in culture to benzo(a)pyrene, benz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but do not display the induction response when treated with 3-methylcholanthrene (MCA), the classical inducer of cytochrome P1-450. Induction of cytochrome P1-450 is regulated by the Ah receptor which initially binds inducing chemicals in the cytoplasm, after which the inducer x receptor complex translocates into the nucleus. Cytosolic and nuclear forms of the Ah receptor can be detected in C3H/1OT1/2 cells using [3H]TCDD as the radioligand in culture, but specific Ah receptor binding is not detectable within C3H/1OT1/2 cells incubated with [3H]MCA. In contrast, in Hepa-1c1 cells, which exhibit cytochrome P1-450 induction when treated with MCA, cytosolic and nuclear Ah receptor can be detected by incubation of the cells either with [3H]MCA or with [3H]TCDD. Nonradioactive MCA is able to compete with [3H]TCDD for Ah receptor sites in C3H/1OT1/2 cells, but the relative potency of MCA as a competitor is lower within C3H/1OT1/2 cells than in C3H/1OT1/2 cytosol during extracellular incubation. Specific binding of [3H]MCA to Ah receptor can be detected by incubation of [3H]MCA with C3H/1OT1/2 cytosol outside the cell. The selective loss of response to MCA as a cytochrome P1-450 inducer (while retaining response to other inducers) appears to be due to defective interaction of MCA with the Ah receptor within the intracellular environment. The specific molecular alteration which makes the MCA x receptor complex ineffective within C3H/1OT1/2 cells is unknown. Some fibroblast lines other than C3H/1OT1/2 also selectively fail to respond to MCA; thus, this variation in Ah receptor function may not be due to a mutational change in the Ah regulatory gene which codes for the Ah receptor.

Cell-free protein synthesis in t,e rabbit liver ribosomal system. II. Large scale preparation of purified 80S ribosomes.

A procedure for the preparation of a large quantity of biologically active, highly purified ribosomes from rabbit liver is described. The method employs polyethylene glycol-dextran sulfate parition and DEAE-cellulose chromatography to overcome the limitations encountered in conventional procedures. The entire process takes only 48 h to obtain 10,000 A(260) units of ribosomes. The ribosomes thus obtained are predominantly 78S particles with a constant protein-RNA ratio of 0.95. The ribosomes are free from RNase, amino-acyl-tRNA synthetase, and amino-acyl-tRNA: protein transferase activity. The protein synthesizing activity is dependent on added mRNA and protein factors. These ribosomes are stable for prolonged periods of storage in a liquid nitrogen refrigerator.