Analysis of cathepsin D in human breast cancer: usefulness of the processed 31 kDa active form of the enzyme as a prognostic indicator in node-negative and node-positive patients.

The relative amounts of the precursor (52 kDa) and processed (31,27 kDa) forms of cathepsin D have been analyzed by Western blotting in biopsied breast tissue cytosols from 134 lesions from invasive breast cancer patients, 24 lesions from patients with ductal carcinoma in situ (DCIS), 227 lesions from benign breast disease patients, and 28 lesions from normal control subjects. The mean relative percentage amount of the 31 kDa form was significantly increased (p < 0.001) in the invasive breast cancer group compared to the other three groups. In addition, the mean relative percentage amount of the 31 kDa form was significantly increased (p < 0.05) in node-positive compared to node-negative breast cancer patients. In the benign breast disease group, patients with proliferative-type disease had a significantly increased (p = 0.02) mean relative percentage amount of the 31 kDa form of cathepsin D compared to patients with nonproliferative-type disease. Invasive breast cancer patients were followed for up to 75 months to determine if the relative percentage amount of the 31 kDa form of cathepsin D was predictive of disease-free and overall survival. Although the amount of the 31 kDa form was not predictive of disease-free survival, patients in the 'high' 31 kDa group (> 18%) were significantly (p < 0.05) more likely to die than patients in the 'low' 31 kDa group (< or = 18%). The 12 patients who died were all node-positive and in the high 31 kDa group. It thus appears that the relative amount of the processed, active 31 kDa form of cathepsin D is a useful prognostic indicator, at least in node-positive breast cancer patients.

Characterization of purified cathepsin D from malignant human breast tissue.

The aspartyl protease cathepsin D (EC 3.4.23.5) appears to be found in increased amounts and/or abnormally secreted in breast cancer cells, and may contribute to the metastatic spread of malignancy. In the present study, cathepsin D was purified 4800-fold in 20% yield from malignant human breast tissue using affinity chromatography on pepstatin-agarose and DEAE-Sephadex chromatography. Slab gel SDS-PAGE of the purified cathepsin D indicated the presence of three major protein bands (31, 13, 12, kDa) and two minor protein bands (47, 29 kDa). Western blotting indicated that the 31 kDa band was the major immunoreactive species. Isoelectric focusing indicated that the purified cathepsin D consisted of three major isoforms at approximate pIs of 7.4, 7.0 and 6.6, and a possible isoform of lower activity centered around pI 3.2. The pH curve of purified cathepsin D indicated a broad optimum centered around pH 3.4. Lectin blotting suggested the presence of mannose residues but no evidence was found for lectin-available sialic acid, fucose, N-acetylglucosamine and galactose residues. The investigated properties of purified cathepsin D from malignant breast tissue are very similar, if not identical, to the properties of cathepsin D previously purified from normal human breast tissue. Our findings suggest that the elevated activity and antigenic levels of cathepsin D in malignant breast tissue are due to increased amounts of apparently normal enzyme.

Purification and characterization of cathepsin D from normal human breast tissue.

The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad area of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.

Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines.

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.

Peptide mapping of the subunits and deglycosylated polypeptides of human liver alpha-L-fucosidase.

The subunits of human liver alpha-L-fucosidase have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, excised, and subjected to peptide mapping after CNBr cleavage or trypsin digestion. The CNBr peptide maps of the glycosylated 56- and 51-kDa subunits were similar except that the larger subunit had several peptides with M(r)s shifted higher than those apparent for the smaller subunit. These M(r) differences were almost completely eliminated when CNBr peptide mapping was performed on the deglycosylated 48- and 45-kDa polypeptides, suggesting that the M(r) differences were due to carbohydrate differences. Minor differences not related to glycosylation were found in the CNBr peptide maps for the 48- and 45-kDa polypeptides including the presence of small amounts of three peptides in the larger polypeptide not found in the smaller polypeptide. Sequence analysis suggested that both the 48- and the 45-kDa polypeptides were blocked at their amino-termini but analysis of the largest CNBr peptide from each polypeptide indicated an identical 13-amino-acid sequence corresponding to residues 6 through 18 from the cDNA-deduced sequence of mature alpha-L-fucosidase. Tryptic peptide mapping indicated very similar HPLC peptide profiles for the deglycosylated 48- and 45-kDa polypeptides except for the presence of small amounts of six peaks present in the larger polypeptide which were not detected in the smaller polypeptide. The overall results provide the first evidence that the polypeptides of human liver fucosidase are very similar and probably encoded by the same gene. However, minor differences in the polypeptides exist, possibly due to normal allelic variation, alternative splicing, proteolytic processing, and/or posttranslational modifications other than those due to glycosylation.

Structural characterization of the N-glycans of a humanized anti-CD18 murine immunoglobulin G.

This study characterized the N-glycans of a humanized immunoglobulin G4 (IgG4) expressed in NS/O mouse myeloma cells and directed against the CD18 family of adhesion-promoting receptors on leukocytes. The N-glycans were released from the purified recombinant IgG by N-glycanase treatment, purified by Sephadex G50 chromatography, and fractionated by Bio-Gel P-4 chromatography into three oligosaccharide pools. Each pool was analyzed individually by glycosyl composition analysis, high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), 600-MHz 1H-NMR spectroscopy, and electrospray-ionization mass spectrometry. In addition, each of the three pools was subfractionated by HPAEC and the isolated subfractions that contained sufficient material were hydrolyzed and analyzed for glycosyl composition by HPAEC-PAD. The overall results indicate the presence of five oligomannoside-type structures (containing 5 to 8 Man residues) which are not usually found in IgG, and the presence of eight diantennary (mostly truncated) N-acetyllactosamine-type structures which are typical of mouse and human IgGs. The N-acetyllactosamine-type structures were heterogeneous with regard to alpha(1-->6) fucosylation of the linkage GlcNAc, and the presence or absence of GlcNAc and/or Gal beta(1-->4)GlcNAc extending the core pentasaccharide (Man3GlcNAc2). No evidence was found for the presence of sialic acid or bisecting GlcNAc residues on the N-acetyllactosamine-type chains. The latter finding suggests that the N-glycans of this humanized IgG are of the mouse type.

Western blotting and enzymatic activity analysis of cathepsin D in breast tissue and sera of patients with breast cancer and benign breast disease and of normal controls.

Increased total antigen amounts of cathepsin D in breast tissue have been reported to be associated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients. In the present study, Western blotting analysis has been used for the first time to determine the relative amounts of precursor and processed forms of cathepsin D in sera and breast tissue of patients with breast cancer, benign breast disease, and normal controls. Sera gave similar blots for breast cancer patients and controls with two major forms of cathepsin D (M(r) 52,000 and 27,000). Malignant breast tissue contained the two forms of cathepsin D found in sera and an additional M(r) 31,000 form which was found in significantly increased (P < 0.001) relative amounts in breast tissue from 43 breast cancer patients [24 +/- 12% (SD)] when compared to 51 benign breast disease patients (13 +/- 8.9%) and 23 normal controls (1.8 +/- 4.4%). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.41) in relative amounts of the M(r) 31,000 form of cathepsin D between proliferative-type and non-proliferative-type fibrocystic breast disease. A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantly increased (P < 0.001) amounts of pepstatin-inhibitable, cathepsin D-specific activity in breast tissue from 36 breast cancer patients (2.2 +/- 1.4 units/mg of protein) when compared to 47 benign breast disease patients (0.63 +/- 0.43) and 23 normal controls (0.24 +/- 0.21). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.21) in pepstatin-inhibitable, cathepsin D-specific activity between proliferative-type and nonproliferative-type fibrocystic breast disease. The positive correlation (r = 0.82) of increased amounts of the M(r) 31,000 form of cathepsin D and increased pepstatin-inhibitable, cathepsin D enzymatic activity in malignant breast tissue suggests that the M(r) 31,000 form is the proteolytically active form of the enzyme which may be involved in the development and/or metastatic spread of breast cancer.

Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast.

The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2,C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.

Evaluation of serum sialic acid and carcinoembryonic antigen for the detection of early-stage colorectal cancer.

Various expressions of elevated serum sialic acid (total sialic acid, TSA: lipid-associated sialic acid, LASA; LASA/TSA; TSA normalized to total protein, TSA/TP) have been evaluated and compared with increased serum carcinoembryonic antigen (CEA) levels for the detection of early-stage colorectal cancer. This evaluation was done blindly on a coded panel of 320 sera from staged colorectal cancer patients and controls provided by the Mayo Clinic--National Cancer Institute Diagnostic Bank. Unlike the findings of a previous preliminary study (Tautu et al., JNCI 80:1333-1337, 1988), the ratio of LASA/TSA was not useful for detecting early-stage (Dukes A and B) colorectal cancer. However, TSA and TSA/TP values were significantly elevated in each colorectal cancer subgroup compared with normal controls. TSA and TSA/TP values displayed a marginally better discriminatory power than CEA values in the case of Dukes A subgroup with respect to normal controls. CEA still appears to be the best single overall marker for discriminating between colorectal cancers and controls. However, multiple marker analysis using CEA and TSA (and related markers) appears to be more sensitive than CEA alone for detecting colorectal cancer.

Serum sialic acid and carcinoembryonic levels in the detection and monitoring of colorectal cancer.

Total sialic acid (TSA), total protein (TP), TSA normalized to total TP (TSA/TP), and carcinoembryonic antigen levels were determined in 146 consecutive colorectal patients. These results were compared with results from 73 people with nonmalignant gastrointestinal disease, and with results from 96 normal controls. All malignancies were staged according to the Astler-Coller modification of Dukes' classification for colorectal cancer. All blood samples were drawn before surgical therapy. The TSA/TP ratio for colorectal cancer was 13.4 (mg/gm) in contrast to 12.1 (mg/gm) for pathologic controls, and 9.7 (mg/gm) for normal controls. Student's t test showed a P value less than 0.001 for normal controls and a P value less than 0.001 for pathologic controls. The TSA/TP also showed statistical significance in Dukes A, B2, C, and D subgroups when compared with normal controls. There were only four patients with stage C1 carcinoma, thus statistical analysis would be questionable. In contrast, carcinoembryonic antigen levels showed no significant elevations until Dukes C2 tumors were encountered. These preliminary findings suggest that TSA/TP ratio may detect colorectal cancer patients with less tumor burden and be more beneficial as a tumor marker than CEA for monitoring patients with colorectal cancer.

Analysis and HPLC fractionation of urine from patients with cystic fibrosis, chronic lung diseases and normal controls.

The amounts of creatinine, protein, carbohydrate and sialic acid in the urine of 19 patients with cystic fibrosis (CF), 12 normal controls and 11 pathological controls with chronic lung disease have been determined. The mean creatinine excretion levels of the total CF group as well as the CF subgroups are significantly decreased when compared to normal controls but comparable to pathological controls. Mean urinary protein levels appear to be increased in patients with CF compared to normal controls and pathological controls but the increased levels resulted from factors (e.g., presence of diabetes mellitus) other than CF. No significant differences were found in amounts of total carbohydrate and sialic acid in urine and fractionated urinary preparations for the total group of nondiabetic patients with CF when compared to both normal and pathological controls. HPLC fractionation of low Mr (less than 10,000 Daltons) urinary preparations indicated the presence of an unknown peak in all of the antibiotic-treated CF patients, 43% of CF patients on low or no medication, 17% of the normal controls and 9% of the pathological controls. The present results illustrate the importance of including appropriate pathological controls and dividing patients with CF into subgroups according to clinical factors and types of therapy employed.

Malignant cell glycoproteins and glycolipids.

The cell surface is involved in cell growth and division, cell-cell interaction, communication, differentiation and migration, and other processes likely to be involved in malignant transformation and/or the metastatic spread of cancer. Although there are many alterations of glycoproteins and glycolipids on the malignant cell surface, it is unclear whether these alterations are epiphenomena or an integral part of the malignancy process. This article reviews the recent literature and some earlier studies relevant for understanding emerging concepts and trends with respect to malignant cell glycoconjugates. Emphasis is on structural alterations of the carbohydrate portions of malignant cell glycoproteins and glycolipids and on the enzymes (glycosyltransferases and glycosidases) involved in their metabolism. Practical applications derived from malignant cell glycoconjugate studies are discussed briefly with respect to the diagnosis, staging, monitoring, and treatment of malignant disease. The review concludes by indicating which research areas on malignant cell glycoconjugates are likely to be fruitful in increasing our basic understanding of, and ability to deal effectively with, malignant disease.

The effect of alkaline borohydride treatment on N-linked carbohydrates of glycoproteins.

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc beta(1-N)Asn with alkali, alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field 1H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing the N-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of approximately 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing the N-linked carbohydrates as oligosaccharide-alditols.

Improved procedure for determination of serum lipid-associated sialic acid: application for early diagnosis of colorectal cancer.

We have developed a simple and reliable procedure for determining serum levels of lipid-associated sialic acid (LASA) in crude preparations. This method extracts essentially all gangliosides, excludes glycoprotein-bound sialic acid, and gives LASA values (0.5-1.0 mg/dL) in good agreement with values for isolated serum gangliosides. The procedure was used to determine serum levels of LASA in patients with colorectal cancer, in patients with nonmalignant diseases (pathological control subjects), and in normal control subjects. The results indicated that the percentages of total sialic acid (TSA) comprised by LASA (LASA/TSA X 100) were elevated in patients with the earliest stages of colorectal cancer, compared with percentages in normal control subjects (P less than .001) and pathological control subjects (P less than .01).

Total and lipid-associated serum sialic acid levels in cancer patients with different primary sites and differing degrees of metastatic involvement.

Serum total sialic acid (TSA) and lipid-associated sialic acid (LASA) levels have drawn considerable interest because of carbohydrate aberrations in malignant cells. The current investigation determined the TSA, LASA, total protein (TP), and TSA/TP values for 171 cancer patients with various primary sites and differing degrees of metastatic disease, 102 patients with nonmalignant diseases (pathologic controls), and 42 normal individuals. Data analysis indicated significant (p less than 0.01) increases in the mean (+/- SD) TSA and TSA/TP values in the cancer patients (78.1 +/- 19.2 mg/dl and 12.4 +/- 3.8 mg/g, respectively) and in the pathologic controls (76.0 +/- 7.5 mg/dl and 11.6 +/- 2.5 mg/g) when compared to the normal controls (67.3 +/- 7.1 mg/dl and 9.0 +/- 1.1 mg/g), and a significant decrease in the mean TP values in the cancer patients (6.4 +/- 1.1 g/dl) and pathologic controls (6.6 +/- 1.1 g/dl) when compared to normal controls (7.5 +/- 0.5 g/dl). No significant difference was observed between groups in LASA values. Further analysis of the data in patient subgroups based on the tissue involved, specific disease, or severity of the malignancy indicated that the lack of specificity of the markers was due primarily to restricted subgroups and that the sensitivity of TSA and TSA/TP increased as the malignancy became more severe. The results show that TSA/TP was the most useful of the markers tested for detecting malignancies. This marker should prove useful for monitoring malignant disease recurrence and/or progression and for evaluating the effectiveness of various therapeutic approaches.

Variant serum beta-hexosaminidase as a biochemical marker of malignancy.

Previous studies have demonstrated a variant form of beta-hexosaminidase in metastatic tumor tissue of human liver and in sera from cancer patients with liver metastases. The current investigation examined sera for the presence of the variant form of beta-hexosaminidase from a large and heterogeneous group of cancer patients (with different primary sites and differing degrees of metastatic involvement), from normal controls and pathological controls with nonmalignant diseases. Comparison of the total serum beta-hexosaminidase activity levels and the percentage of the total activity comprised of beta-hexosaminidase B (Hex B) revealed no significant differences (P greater than 0.01) between the three groups. Analytical isoelectric focusing indicated that the variant beta-hexosaminidase was present in 80% of 108 cancer patients, 37% of 27 pathological controls and 11% of 18 normal controls. Examination of subgroups of the cancer patients based on the extent of metastasis revealed that there was a significant increase in total serum beta-hexosaminidase activity and the presence of variant beta-hexosaminidase in the sera as the disease progressed. These results suggest that serum beta-hexosaminidase may be a useful marker for monitoring the progression of malignant disease.

beta-Hexosaminidase from colon and sera of dukes-classified colorectal cancer patients: activity levels, isozyme patterns, and kinetic properties.

Total activity levels, isozyme patterns, and kinetic properties of beta-hexosaminidase were studied in crude supernatants of malignant and adjacent, uninvolved normal colon tissues and in sera from 28 Dukes-classified colorectal cancer patients. A significant increase (P less than .001) in both beta-hexosaminidase activity and beta-hexosaminidase specific activity and a significant increase (P less than .01) in the relative percentage of activity comprised by the basic thermostable form of beta-hexosaminidase (Hex B) isozyme were found in malignant tissue compared to the activities seen in uninvolved normal colon tissue. No apparent correlations were found between either beta-hexosaminidase activity levels or relative percentage of Hex B and Dukes category. Kinetic analysis indicated that the thermolabile form of beta-hexosaminidase and Hex B from malignant colon were comparable to the corresponding isozymes from normal colon with regard to thermostability after preincubation at 50 degrees C and pH activity curves (optimum between pH 4.0 and 5.0). Significantly decreased (P less than .05) beta-hexosaminidase activity was found in sera of the 28 colorectal cancer patients (17.3 +/- 5.2 U/ml, mean +/- SD) when compared to the activity in 19 controls with nonmalignant diseases (21.4 +/- 8.2 U/ml) and 17 normal controls (21.3 +/- 6.4 U/ml). Isoelectric focusing indicated that a peak of beta-hexosaminidase activity with an isoelectric point value (9.5) comparable to that of the peak found in increased amounts in malignant colon was detectable in the sera of 36% of the colorectal cancer patients and 11% of the controls.

Isoelectric focusing of alpha-L-fucosidase from human embryonal carcinoma.

Isoelectric focusing profiles of alpha-L-fucosidase recovered from human embryonal carcinoma (EC) and seminoma were compared with those of mouse germ cell tumor-derived stem cells and some human somatic cell neoplasms in an attempt to determine whether EC cells could be used as a source of human basic isoelectric forms of the enzyme previously identified in normal and malignant mouse embryonic cells. alpha-L-Fucosidase activity in all human tumors was associated with isoelectric forms in the isoelectric point (pl) range between approximately 4.5 and 7, corresponding to the range seen in normal human tissues. The basic isoelectric forms (pl values, 7.5-9.5) that predominate in the embryonic isoelectric focusing pattern in mouse were not found in human neoplasms. The present data illustrate another difference between human and mouse EC and show that mouse EC cells are not a complete replica of equivalent human tumor cells.