Alterations in cathepsin H activity and protein patterns in human colorectal carcinomas.

Our analyses of cathepsin H activity levels and protein forms in human colorectal cancers compared to matched control mucosa support the concept that altered proteinase expression patterns may reflect both cancer stage and site. Cathepsin H-specific activity was significantly increased in colorectal cancers compared to control mucosa (P = 0.003; n = 77). Highest specific activities and cancer/normal ratios (C/N) for activity were measured in Dukes' B and C stage carcinomas, cancers involved in local spread and invasion to lymph nodes. In contrast, cathepsin B and L activities analysed in the same paired extracts had been shown to be most frequently elevated in earlier stage carcinomas (Dukes' A and B), confirming that cathepsin H demonstrates a distinct pattern of expression during colorectal cancer progression. Although cathepsin H activities were most commonly elevated in Dukes' C cancers at all colon sites, both specific activity and C/N ratios were significantly higher for cancers of the left colon compared to other colon locations. A subset of 43 paired extracts analysed on Western blots also revealed consistent changes in cathepsin H protein forms in cancers. Normal mucosa typically showed a strong protein doublet at 31 and 29 kDa while cancers demonstrated decreased expression or total loss of the 31 kDa protein (90% of cases), equal or increased expression of the 29-kDa protein (67% of cases) and the new appearance or up-regulation of a cathepsin H band at 22 kDa (78% of cases). C/N ratios for cathepsin H enzyme activity correlated significantly with C/N ratios for the 29 kDa mature single-chain protein form (P < 0.001), with increased activity most commonly associated with elevated expression of 29-kDa cathepsin H but also with up-regulation of the 22-kDa band, suggesting a shift to more fully processed, mature active cathepsin H protein forms in cancers. Changes in cathepsin H expression were also detected by immunohistochemistry as elevated cathepsin H staining in tumour epithelial cells.

Cathepsin B activity and protein levels in thyroid carcinoma, Graves' disease, and multinodular goiters.

Cathepsin B (CB) is involved in the hydrolysis of thyroglobulin (Tg) and thought to be regulated by thyroid stimulating hormone (TSH) in the normal thyroid. Our analyses of 91 thyroid tissues from 71 patients with Graves' disease (GD), multinodular goiter (MNG), papillary carcinoma (PC), or follicular carcinoma (FC), demonstrated a 2-fold increase in expression of CB in GD and an average increase of 1.5-fold in MNG (varying from 10-fold below normal to 6-fold above normal in MNG nodules), as might be predicted by the altered functional status of thyroid follicular cells in those diseases. However, CB activity was not downregulated in conjunction with the known "blocking effect" of malignancy on many thyroid functions, but rather increased on average 9-fold in papillary carcinomas (n = 33), and also showed a marked increase in 2 follicular carcinomas. Activity measurements were confirmed by CB protein detection on Western blot with moderately increased CB protein levels demonstrated in GD, variable expression in nodules of MNG, and markedly increased protein expression in carcinomas. In all diseased states, increased protein was detected primarily as overexpression of the 27 kd heavy chain of 2-chain mature CB and less frequently as overexpression of 31 kd single-chain mature CB. However, an additional 35 kd protein form was noted in 3 of 9 PCs, 1 of 2 FCs, and 1 of 4 GD cases but in none of 10 MNG cases. In conjunction with elevated CB activity plus additional protein bands on Western blots, altered patterns of CB immunohistochemical staining were observed, irrespective of the type of thyroid disease, suggesting certain common changes in CB expression, posttranslational processing, and vesicular trafficking. In summary, GD and MNG thyroid tissues demonstrated altered CB expression in keeping with predicted functional changes in thyroid follicular cells, while increased CB expression in carcinomas indicated a more pathological role for CB in thyroid cancers, possibly related to the processes of invasion or metastasis.

Presence of activated ras correlates with increased cysteine proteinase activities in human colorectal carcinomas.

The metastatic potential of ras-transfected cells has been attributed in part to significant ras induction of proteinase expression. To determine whether primary cancers also demonstrate higher cysteine proteinase activities in the presence of activated ras genes or altered ras protein expression, we have analyzed 60 primary human colorectal carcinomas for ras gene or protein changes together with the expression of cathepsins B and L. Cancers containing K-ras mutations (47% of 60 carcinomas) demonstrated greater increases in cathepsin L activity than cancers without K-ras mutations (p = 0.029), with particularly significant correlations for earlier stage cancers (Dukes' A and B carcinomas, p = 0.006). Western blots used to characterize ras protein patterns in the same cancer/normal pairs have demonstrated that N-ras protein is more highly expressed in colon tissues than H- or K-ras proteins and that N-ras overexpression occurs in almost 70% of colorectal cancers, with or without a concurrent change in electrophoretic mobility of N-ras protein. Our current study has now shown that N-ras protein overexpression alone does not significantly induce cathepsin B or L activity levels in colon cancers. However, carcinomas demonstrating altered N-ras protein forms, in the absence of any K- or N-ras mutations, expressed significantly higher levels of cathepsin B and L activities compared with carcinomas with normal N-ras protein banding patterns. Our data suggest that colorectal carcinomas with either K-ras mutations or altered forms of N-ras protein may increase their tumorigenic potential via the induction of cathepsin L or B expression levels. Our results also confirm that ras oncogene up-regulation of cathepsin B and L activities, previously reported in cultured cells, is a frequent event in primary human colorectal carcinomas.

Elevations in cathepsin B protein content and enzyme activity occur independently of glycosylation during colorectal tumor progression.

Western blots, enzyme assays, protein glycosylation studies, and immunohistochemical staining were used to characterize cathepsin B expression at successive stages of colorectal tumor progression. In normal colon mucosa and premalignant adenomas, cathepsin B expression was predominantly due to mature two-chain protein detected on Western blots as the nonglycosylated 27-kDa form, with overexpression of this protein occurring in only 4 of 18 adenomas. Overexpression increased significantly in Dukes A and B carcinomas (26 of 37 cases), with cathepsin B protein generally detectable in carcinomas as a combination of both 27-kDa nonglycosylated and 28-kDa glycosylated mature two-chain forms. Glycosylated cathepsin B protein in carcinoma extracts was sensitive to PNGase F but resistant to Endo H, indicating a pattern consistent with complex rather than high mannose type glycosylation. When sorted by advancing tumor stage, peak expression of cathepsin B protein occurred in carcinomas involved in local invasion compared with adenomas or metastatic cancers. At all stages, cathepsin B activity correlated significantly with the levels of heavy chain mature cathepsin B protein (r = 0.6682, p < 0.0001) irrespective of glycosylation. Immunohistochemical staining of cathepsin B protein revealed fine diffuse cytoplasmic staining in both adenomas and carcinomas compared with coarse granular cytoplasmic staining (typical of lysosomes) seen in matched normal mucosa. Our results demonstrate several sequential, apparently independent changes in cathepsin B expression during colorectal tumor progression including early changes in subcellular localization, up-regulation of cathepsin B protein and activity in invasive cancers, and altered protein glycosylation detected in malignant tumors at all stages.

Characterizing human colorectal carcinomas by proteolytic profile.

Clinicopathologic staging of colorectal cancers cannot always predict aggressiveness of prognosis for a particular patient. We have used activity assays for cysteine proteinases, cathepsins B, L and H (CB, CL and CH) and matrix metalloproteinases, MMP-2 and MMP-9, to identify several distinctive, reproducible proteolytic profiles in a large set of colorectal carcinomas. We observed that individual proteinases demonstrated specific and distinct levels of activity at different cancer stages, possible reflecting non-random steps in a proteolytic cascade related to tumor development. We also observed that individual colon cancers fell into relatively few categories when characterized for the combined expression of three proteinases: CB, CL and MMP-9. Four proteolytic profiles, designated "Early", "Middle", "Late", and "High", could be used to define almost 80% of the colorectal carcinomas analyzed. Such profiles, based on the expression of several proteinases in a given tumor, provided information independent of clinical stage and may identify crucial variations in tumor behavior.

N-ras protein: frequent quantitative and qualitative changes occur in human colorectal carcinomas.

Point mutation and overexpression are recognized mechanisms for ras activation in malignancy. However, little information is available on overexpression of N-ras protein compared with H- or K-ras proteins, as N-ras-specific antibodies have only recently become available. For comparative analyses of ras protein levels, we have probed Western blots of extracts from 9 normal human tissues and 55 pairs of colorectal carcinoma and matched control mucosa, using monoclonal antibodies (MAbs) specific for H-, K- or N-ras proteins. On multi-tissue blots, N-ras protein was more highly expressed in colon than in the other human tissues analyzed, suggesting a role for N-ras in colorectal function. N-ras protein levels in multiple independent extracts of normal colon mucosa were consistently higher than either K-ras protein or H-ras protein levels. In 69% of colon carcinomas, N-ras protein levels were increased an average of 4.8-fold over normal mucosa. Overexpression of K-ras protein was also observed in colon cancers but less frequently (13% of cases) than N-ras protein. H-ras protein levels were too low for comparative studies. Alterations in N-ras protein mobility, possibly reflecting increased post-translational processing, were also detected in 42% of colon carcinomas. N-ras protein, typically present as a single 23 kDa band in normal mucosa, was expressed in some cancers as a 22 kDa band or as multiple bands of 20-23 kDa. Sequencing of N-ras DNA from 6 carcinomas with these variations in protein mobility did not reveal mutations in codons 12, 13, 59 or 61. Thus, frequent quantitative and qualitative changes in N-ras protein expression, which do not appear to correlate with the presence of typical N-ras point mutations, result in abnormal N-ras protein patterns in human colorectal carcinomas.

Marked increases in cathepsin B and L activities distinguish papillary carcinoma of the thyroid from normal thyroid or thyroid with non-neoplastic disease.

Cathepsin B (CB) and cathepsin L (CL) are cysteine endopeptidases involved in the processing of thyroglobulin (Tg) in the normal thyroid. As thyroglobulin expression is frequently altered in thyroid carcinomas, we have analyzed 42 human thyroid tissues from 40 patients to study the effect of malignant transformation an the expression of these endopeptidases. Our samples included 18 cases of papillary carcinoma (of which 10 also had matched adjacent normal thyroid tissue), 6 cases of normal thyroid from autopsy patients, 1 case of follicular carcinoma, 2 cases of medullary carcinoma, 2 cases of follicular adenoma, 3 cases of Hashimoto's thyroiditis (HT) and 10 samples from 8 patients with multi-nodular goiter (MNG). Enzyme-specific activities were increased 15-fold for CB and 9-fold for CL in papillary carcinoma compared with normal adjacent thyroid tissue or normal thyroid from autopsies. CB mRNA content was also markedly increased in papillary carcinoma compared with normal thyroid, primarily due to elevated levels of the 2.2-kb CB mRNA transcript. In thyroids with nonneoplastic diseases, including MNG and HT, there was no significant increase in either CB or CL enzyme activities nor CB mRNA levels compared with normal thyroids from non-cancer cases. Immunohistochemical studies on papillary carcinomas revealed increased CB staining in papillary carcinoma cells, with prominent staining close to the basement membranes of many of the neoplastic cells. Our observations suggest that CB and CL enzyme activities are potentially useful new biochemical markers for distinguishing papillary carcinoma of the thyroid from non-neoplastic thyroid disease.

Detection of p21ras mutations in colorectal adenomas and carcinomas by enzyme-linked immunosorbent assay.

BACKGROUND: Point mutations of the ras protooncogene, primarily within codons 12 and 13, are commonly identified in colorectal carcinomas and large adenomas. Despite data suggesting that ras genotyping may have clinical significance with respect to colorectal cancer screening and prognosis, more widespread use has been limited because of the lack of a suitable assay system. The principal objective of this study was to assess the feasibility and validity of a qualitative enzyme-linked immunosorbent assay (ELISA) for detecting the four most common ras mutations in human colorectal tumors at the protein (p21ras) level. METHODS: Tissue homogenates (11-121 micrograms) from endoscopically or surgically resected colorectal adenomas, carcinomas, and normal mucosae were evaluated by a commercially available ELISA (Oncogene Science, Inc. Cambridge, MA) for mutant p21ras containing arginine position 12 (arg12), valine position 12 (val12), aspartate position 12 (asp12), and aspartate position 13 (asp13) amino acid substitutions. Portions of the same tissue from an initial series of 27 specimens also were subjected to mutant-enriched polymerase chain reaction (PCR) and/or PCR amplification with subsequent DNA sequence analysis to validate the ELISA data. RESULTS: Forty-seven adenomas, 9 carcinomas, and 14 normal mucosae were assayed. Mutations were identified in 16 (34%) of the adenomas (7-asp12, 7-val12, 2-asp13), 3 (33%) of the carcinomas (1-asp12, 1-arg12, 1-asp13), and none of the normal mucosae by ELISA: Polymerase Chain Reaction and DNA sequencing analyses demonstrated identical results for 21 of the 23 (91%) and 14 of 16 (88%) homogenates tested, respectively. The ELISA demonstrated an overall sensitivity of 80-86%, specificity of 90-92%, positive predictive value of 86-100%, and negative predictive value of 86-91%. CONCLUSIONS: The ELISA is a feasible and valid approach for identifying p21ras mutations in human colorectal adenomas and carcinomas.

Human cathepsin B-encoding cDNAs: sequence variations in the 3'-untranslated region.

We have isolated two cathepsin B (CTSB)-encoding cDNAs, hCBF1 and hCBF2, from a normal human embryonic fibroblast library. These clones demonstrate 98% identity to overlapping regions of published human hepatoma and kidney CTSB cDNAs, but show some interesting differences from the published sequences in the 3'-untranslated region (3'-UTR). Both hCBF1 and hCBF2 contain a 10-bp insertion in the 3'-UTR that may permit formation of a highly stable stem-loop structure not present in mRNAs without this insertion. Our hCBF1 cDNA also contains a 1019-bp extension of the 3'-UTR sequence that resembles the long 3'-UTR reported for murine CTSB cDNAs. Probes unique to this 3'-UTR extension hybridize to 4.0- and 1.7-kb CTSB RNAs on Northern blots, but not to the major 2.2-kb mRNA transcript. Our data reveal variations in normal human CTSB transcripts that result from differences in the length of the 3'-UTR, as well as the presence or absence of a stem-loop stabilizing sequence.

Cysteine endopeptidase activity levels in normal human tissues, colorectal adenomas and carcinomas.

We have assayed cysteine endopeptidase activities in 17 types of normal human tissue and in matched sets of colorectal mucosa, adenoma and carcinoma samples. Our data indicate that cathepsin B enzyme levels vary 70-fold and cathepsin L enzyme levels vary 20-fold from one normal tissue to another. Cathepsin B specific activity in normal tissues fell into 3 categories. High activity, with a mean of 156.7 +/- 41.5 nmoles min-1 mg-1 protein, was measured in liver, thyroid, kidney and spleen; intermediate activity, with a mean of 60.2 +/- 8.3 nmoles min-1 mg-1 protein, was measured in heart, colon, adrenal and lung; and low activity, with a mean of 18.4 +/- 9.7 nmoles min-1 mg-1 protein, was measured in prostate, testis, nerve, stomach, pancreas, brain, skeletal muscle, skin and breast. Cathepsin L specific activity fell into 2 categories. High activity, with a mean of 51.1 +/- 4.9 nmoles min-1 mg-1 protein, was measured in thyroid, liver and kidney; and low activity, with a mean of 11.4 +/- 5.5 nmoles min-1 mg-1 protein, was measured in spleen, colon, heart, adrenal, lung, testis, brain, nerve, skin, stomach, pancreas, skeletal muscle, prostate and breast. Our characterization of these enzyme levels provides a reference standard for normal cathepsin B and L activities in human tissues that should enhance the detection of their deregulation in disease states. For example, in studies of colorectal carcinoma and normal mucosa, we observed a significant tumor-specific increase in cathepsin B and L activities with particularly high activity levels in earlier (Dukes' A and B) compared to later (Dukes' C and D) stages of colorectal cancer. In contrast, adenomas from colorectal cancer patients expressed normal levels of cathepsin B activity, providing evidence that the increase in expression of cathepsin B may be a sensitive marker for progression from the pre-malignant to the malignant state in the development of colorectal cancer.

Stage-specific increases in cathepsin B messenger RNA content in human colorectal carcinoma.

Cathepsin B mRNA levels and banding patterns were characterized in human colorectal mucosa and carcinoma tissues from patients with tumors of different Dukes' stages. Quantitation of mRNA content using slot blot hybridization demonstrated an increase in cathepsin B message in seven of eight tumor tissues with an average increase of 3.5-fold over patient-matched control mucosa samples. This tumor-specific increase in cathepsin B mRNA confirms and extends our previous observation that cathepsin B enzyme specific activity levels are significantly elevated in colorectal carcinomas. In fact, the increase in mRNA levels is greater and more consistent than the observed increase in enzyme activity, suggesting that posttranscriptional or posttranslational regulation of cathepsin B expression occurs in colorectal tumors. The mRNA data also support our earlier observation that cathepsin B enzyme activity levels are inversely correlated with Dukes' stage. The tumor-specific increase in cathepsin B mRNA content is almost 4 times greater in earlier stage (Dukes' A and B) tumors than in later stage (Dukes' C and D) tumors. Thus, increased cathepsin B gene expression is particularly characteristic of tumors which are in the process of invading the bowel wall or local tissues compared with tumors which have already spread to more distant sites. Northern blot data on cancer/normal pairs indicate that the increase in cathepsin B mRNA in colorectal carcinoma is due primarily to changes in the amount of the 2.2- and 4.0-kilobase transcripts which are seen in control tissue. However, low levels of two additional cathepsin B mRNA transcripts (1.5 and 3 kilobases in size) were also observed in tumor tissue.

Cysteine protease activities and tumor development in human colorectal carcinoma.

Many studies of malignant cells or tissues in culture have implicated cysteine proteases in the progression of malignancy. We have extended these observations by measuring quantitative and qualitative changes in the expression of cathepsin B-like and L-like cysteine proteases during the growth and development of human colorectal carcinomas. Data derived from matched pairs of normal colorectal mucosa and carcinoma tissue from 27 patients demonstrated that both cathepsin B-like and cathepsin L-like specific activities were significantly elevated (P less than 0.005) in the carcinoma tissue, while levels of endogenous cysteine protease inhibitor remained constant. Correlation of cathepsin enzyme activities with different stages of colorectal cancer demonstrated significantly higher cysteine protease activities in individuals with Dukes' A tumors (tumors confined to the bowel wall) than in patients with more advanced tumors (Dukes' B, C, or D tumors) (P less than 0.01-0.05). The relative proportion of activities contained in tumor epithelial and stromal elements remains to be elucidated. These results suggest an important role for cysteine proteases in the early progression of human colorectal carcinoma.

Expression and induction of cathepsins B and D in K562 cells.

Three distinct lysosomal protease activities have been identified in the human leukemia cell line, K562. These include cathepsin D, the classic protease of the mature red blood cell, as well as two proteases, cathepsins B and H, which have been associated with development and differentiation in a variety of tissues. Each of these three lysosomal proteases was expressed in a specific fashion during hemoglobin induction in K562 cells. Both cathepsin B and cathepsin D activities could be induced by growth of K562 cells in medium containing either hemin or heat-treated serum or by increasing the concentrations of untreated serum in the medium. Cathepsin H activity in the same cells remained unchanged. This is the first report of inducible protease activities in K562 cells. Our identification of specific well-characterized protease activities that change differentially during K562 induction provides a framework for additional studies on the role of proteases in hematopoietic differentiation.

Chloride transport properties of human leukemic cell lines K562 and HL60.

The Cl- transport characteristics of the human leukemic cell lines K562 and HL60, with erythroid and granulocytic phenotypic features, respectively, were investigated. Cl- effluxes were measured with 36Cl- under equilibrium conditions in both cell lines and were found to be three orders of magnitude slower than the unidirectional efflux of Cl- in normal erythrocytes. Induction of differentiation of the K562 cell line with hemin does not affect the rate of Cl- transport, while induction of the HL60 cell line with dimethyl sulfoxide results in a small decrease in the rate of Cl- transport. Cl- transport in both cell lines could be divided into two components. One component is inhibited by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), displays counter-transport characteristics, and has a high energy of activation--all properties characteristic of the human erythrocyte-facilitated anion exchange system. The second component is insensitive to DIDS, is partially inhibited by furosemide, and has a low energy of activation.

Studies of BFU-E in flask cultures of human peripheral and cord blood cells.

Early erythroid progenitors (the burst-forming units-erythroid or BFU-E) from human peripheral and cord blood mononuclear cells were maintained in flask culture for 2 weeks without added erythropoietin (Epo) or erythroid potentiating activity (EPA). These cultures did not develop adherent cell layers and did not support the more mature erythroid colony-forming unit (CFU-E). Samples removed at intervals from these flask cultures were assayed for BFU-E recovery in a plasma clot system in response to a range of Epo doses and to added EPA with time in flask culture. The BFU-E recovered showed increased proliferative capacity and decreased responsiveness to Epo and EPA. These results indicate selection of more primitive erythroid progenitor cells under the conditions described. Peripheral and cord blood mononuclear cell cultures provide a flexible and accessible approach to in vitro studies of human erythropoiesis. Comparative studies with long-term marrow cultures should help to elucidate the role of adherent cells and humoral factors in erythroid differentiation.

Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562.

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3-4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3-5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Cloning and nucleotide sequence analysis of human embryonic zeta-globin cDNA.

Clones of human embryonic alpha-like zeta-globin cDNA were isolated, by detection using cross-hybridization to human alpha-globin cDNA probes, from a cDNA library derived from the mRNA of the human erythroleukemia cell line K562. Nucleotide sequence analysis of these cDNA clones revealed a coding sequence that corresponds perfectly to the independently derived amino acid sequence of the human zeta-globin chain. Comparison of the nucleotide sequence of human zeta-globin cDNA with that of human alpha-globin cDNA confirmed previous estimates of very distant evolutionary divergence between the human zeta- and alpha-globin genes. Nevertheless, the human zeta-globin cDNA sequence shares a remarkable similarity to that of the alpha-globin gene in its codon usage, high G + C base composition, and lack of bias against usage of CG dinucleotides.

Embryonic-fetal erythroid characteristics of a human leukemic cell line.

We have studied a number of cell surface, enzyme, and protein markers in the human leukemic K562 cell line. We have confirmed previous observations that these cells accumulate human embryonic hemoglobins after exposure to hemin. In addition, our results demonstrated that these cells possess in the "ininduced" state i surface antigen, lactate dehydrogenase isoenzymes characteristic of embryonic or fetal erythroid cells, fetal and embryonic globin chains, and globin mRNAs. The levels if i antigen, embryonic globin chains, and embryonic globin mRNA increased substantially after exposure of the cells to hemin in suspension culture. In contrast, K562 cells lacked several surface, enzymatic, and functional properties typical of granulocytes, lymphocytes, monocytes, or adult erythroblasts, including HLA surface antigens, surface immunoglobulins, sheep erythrocyte rosetting, phagocytosis, terminal deoxynucleotidyl transferase, carbonic anhydrase, ABO and Rh blood groups, and adult hemoglobins. The K562 cell line therefore exhibits phenotypic properties of embryonic erythroid progenitor cells and a quantitative increase in the expression of some of these properties can be achieved by exposure of the cells to hemin.