Biomedical subjects

S Shuja

Publications and source records attributed to S Shuja.

10 recordsLinked to original sources

PubMed2000

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.

Adult↗

PubMed1999

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.

Blotting, Western↗

PubMed1998

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.

Blotting, Western↗

PubMed1997

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.

Adenoma↗

PubMed1997

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.

Antibodies, Monoclonal↗

PubMed1996

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.

Adenocarcinoma, Follicular↗

PubMed1991

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.

Adenoma↗

PubMed1991

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.

Biomarkers, Tumor↗

PubMed1989

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.

Adenocarcinoma, Mucinous↗

PubMedDate not supplied

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.

Biomarkers, Tumor↗