Cysteine
proteinases cathepsins (Cats) B and L and their endogenous inhibitors
stefins (Stefs) A and B are implicated in the processes of local and
metastatic tumor spread. They were identified as ...potential
prognosticators in various malignant diseases, particularly in breast
cancer. The aim of the present study was to determine the
concentrations of Cats B and L and Stefs A and B in the tumor and
adjacent normal tissue samples collected from 49 patients (the present
group) with squamous cell carcinoma of the head and neck (SCCHN), using
quantitative immunosorbent assays (ELISA; KRKA d.d., Novo mesto,
Slovenia). Their clinical significance was compared with that from a
previous study (the reference group, 45 patients; Budihna et
al. , Biol. Chem. Hoppe-Seyler, 377: 385–390,
1996). The follow-up of patients from the latter report was updated for
this purpose.
In the present group, significantly higher concentrations of Cat B
( P < 0.0001), Cat L ( P <
0.0001) and Stef A ( P = 0.006) were found in tumors
compared with concentrations in their normal tissue counterparts. Cat
concentrations in normal laryngeal tissue were significantly/marginally
elevated compared with nonlaryngeal tissue (Cat B,
P = 0.02; Cat L, P = 0.06). The
tumor concentration of Cat L was found to correlate with pT
classification ( P = 0.005) and
tumor-node-metastasis stage ( P = 0.05),
whereas the concentrations of Stefs A and B correlated with pN
classification ( P = 0.007 and P = 0.03, respectively) and tumor-node-metastasis stage of the
disease ( P = 0.02 and P = 0.03,
respectively). There was no statistically significant difference
between low and high Cat B or Cat L groups, regarding either
disease-free survival or disease-specific survival, using a
minimum P approach to determine cutoff concentrations.
The risk of disease recurrence and SCCHN-related death was
significantly higher in patients with low Stef A ( P = 0.0006 and P = 0.0005, respectively) and Stef B
( P = 0.0009 and P = 0.0007,
respectively) tumors, compared with those with high-Stef A and Stef B
tumors. These results remained significant even after P s
were adjusted for a possible bias in the estimated effect on survival.
The survival analysis in the reference group also confirmed these
findings (Stef A: P = 0.0009 and
P = 0.002, respectively; Stef B:
P = 0.03 and P = 0.009,
respectively). To avoid any possible bias arising from the differences
between the laboratories that performed the biochemical analysis, the
concentrations of both Stefs in the present group and in the reference
group were standardized and coupled together to form a uniform group.
In univariate survival analysis, standardized values of Stef A and Stef
B correlated inversely with the rate of relapse ( P = 0.0000) and mortality rate ( P = 0.0000).
Multivariate regression analysis showed that the standardized value of
Stef A is the strongest independent prognostic factor for both
disease-free survival and disease-specific survival. These findings
show the specific role of Cats B and L and Stefs A and B in the
invasive behavior of SCCHN. Furthermore, Stef A proved to be a reliable
prognosticator of the risk of relapse and death in patients with this
type of cancer.
Gliomas are the most common form of intrinsic primary brain tumors, that extensively invade the surrounding normal brain tissue. The failure of chemotherapy treatment of these tumors is chiefly ...attributed to drug-resistance. From human glioblastoma we developed two cell sublines resistant to cisplatin due to acute (AT cells) or continuous (CT cells) treatment with clinically relevant doses of cisplatin. We examined their sensitivity to different cytostatics by colorimetric MTT assay. The concentrations of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were determined by the ELISA assay. The results reveal that both AT and CT cells became resistant to cisplatin and vincristine; AT cells became resistant also to etoposide. Both AT and CT cells did not significantly change their sensitivity to doxorubicin, 5-fluorouracil and chlorambucil. Concentrations of uPA and PAI-1 were increased in CT cells, with no change in AT cells. In the conditioned medium of both, AT and CT cells, the level of uPA were increased. No differences in concentrations of PAI-1 in the conditioned medium of these cells were found. Thus, our results show that drug-resistance of glioblastoma cells may be accompanied with the increased levels of markers for tumor invasion.
A chimeric mouse-human antibody has been created that recognizes an antigen found on breast cancer cells and melanoma cells. Immunoglobulin constant domains of mouse monoclonal antibody CDI 315B Cγ1 ...and Ckappa, were substituted by the human Cγ1 and Ckappa. The CDI 315B variable heavy and light chain regions were PCR amplified from hybridoma RNA and sequenced. Mouse variable VH and VL regions were joint to human IgG1 and kappa constant regions and subcloned into pcDNA3 expression vectors. The Sp2/0 murine myeloma cells were transfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of chimeric antibodies was tested by indirect ELISA using B16F1 murine melanoma cells as well as MCF7 human breast cancer cells, as antigen.
In cytosols of tumour and normal tissue of 53 patients suffering from head and neck carcinoma cathepsins D, B, H and L were measured using quantitative immunoreactive assays (ELISA). The values of ...cathepsins D, B and L were significantly higher in tumour tissue, whereas cathepsin H concentration was lower in tumour than in normal tissue. Median cathepsin D values were 27 pmol (tumour tissue) vs. 12 pmol (normal tissue) per mg of total protein, median cathepsin B values were 1.25 micrograms/mg (tumour tissue) vs. 0.23 micrograms/mg (normal tissue) and median cathepsin L values were 39.8 ng/mg (tumour tissue) vs. 20.0 ng/mg (normal tissue). Median cathepsin H values were 1.05 micrograms/mg and 2.20 micrograms/mg for tumour and normal tissue, respectively. Additionally, stefin A and stefin B were measured in tumour and normal tissue samples. In contrast to the cathepsins, the concentrations of these inhibitors of cysteine proteinases was not significantly different between tumour and normal samples. The concentrations of cathepsins D, B, H and L and stefins A and B measured in head and neck tumours, were independent of standard clinical and histological prognostic factors. Significant correlation of tumour tissue values was observed between cathepsins B and L and between both stefins.
Aspartic proteinase cathepsin D (CD) is believed to be associated with proteolytic processes leading to local invasion and seeding of tumour cells. To estimate a potential prognostic value of ...cathepsin D in squamous cell carcinoma of the head and neck, its total concentration was measured immunoradiometrically (ELSA-CATH-D kit, CIS bio international) in cytosols of tumour and adjacent normal tissue samples from 111 patients; in 42/111 patients, the CD concentration was determined in serum samples obtained at diagnosis (serum no. 1) and after the therapy (serum no. 2) from each of these patients. Sera of 15 healthy volunteers served as controls. A significantly elevated concentration of CD was measured in tumour cytosols as compared to normal tissue cytosols (31.1 versus 12.6 pmol/mgp,
P<0.0001) and in cytosols of normal laryngeal tissue than of the oral cavity or pharynx (13.3 versus 11.2 pmol/mgp,
P=0.03). The higher CD tumour concentration correlated with the age of the patients (≤60 versus >60 years, 28.8 versus 32.8 pmol/mgp,
P=0.045) and histopathological tumour grade (G
1+2 versus G
3, 32.6 versus 24.4 pmol/mgp,
P=0.02). In serum samples, a lower concentration of CD was measured in the control group than in the patients (3.6 versus 4.1 pmol/mls,
P=0.045) and in serum no. 1 than in serum no. 2 (4.1 versus 5.1 pmol/mls,
P=0.05). The CD concentration in sera obtained at diagnosis was stage-dependent (S
I–III versus S
IV, 3.9 versus 4.7 pmol/mls,
P=0.09); there was a trend towards lower CD concentrations with an increasing time delay in serum no. 2 sampling (
R
S=−0.20,
P=0.21). No correlation was observed between cytosolic and serum concentrations of CD. We conclude that our results confirm a specific role of CD in the process of invasion and metastasis of squamous cell carcinoma of the head and neck, which might also be of prognostic value in this particular cancer type.
A chimeric mouse-human antibody has been created that recognizes an antigen found on breast cancer cells and melanoma cells. Immunoglobulin constant domains of mouse monoclonal antibody CDI 315B Cγ1 ...and Cκ, were substituted by the human Cγ1 and Cκ. The CDI 315B variable heavy and light chain regions were PCR amplified from hybridoma RNA and sequenced. Mouse variable V
and V
regions were joint to human IgG
and κ constant regions and subcloned into pcDNA3 expression vectors. The Sp2/0 murine myeloma cells were transfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of chimeric antibodies was tested by indirect ELISA using B16F1 murine melanoma cells as well as MCF7 human breast cancer cells, as antigen.
To determine the activity of glutathione (GSH) and concentrations of glutathione S-transferases (GST), urokinase type plasminogen activator (uPA), and plasminogen activator inhibitor type 1 (PAI-1), ...and to evaluate their diagnostic and prognostic value and possible correlation with clinical and histopathological prognostic factors for ovarian carcinomas.
The concentrations of GSH, uPA, PAI-1, and activity of GST were analyzed in 35 tissue samples taken from 10 normal ovaries, 10 benign, 10 primary malignant, and 5 metastatic ovarian tumors. The GSH level and GST activity were determined by spectrophotometric methods, and uPA and PAI-1 concentrations by ELISA commercial kits.
GSH concentrations were significantly higher in primary malignant (126.3+/-12.8 nmol/mg protein) and metastatic (160.5+/-24.3 nmol/mg protein) ovarian tumor specimens than in normal ovarian tissue (48.9+/-8.1 nmol/mg protein, p<0.003 for both carcinoma groups) or benign ovarian tumor samples (35.2+/-5.0 nmol/mg protein, p=0.001). The GST activity was significantly higher in primary malignant (245.8+/-22.7 nmol/min/mg protein) and metastatic (303.7+/-48.8 nmol/min/mg protein) ovarian tumor tissues than in benign tumor specimens (105.9+/-16.2 nmol/min/mg protein, p<0.004 for both carcinoma groups) or normal ovarian tissue samples (133.2+/-32.0 nmol/min/mg protein, p<0.044 for both carcinoma groups). There were no statistical differences in uPA and PAI-1 concentrations between normal, benign, and malignant tumor samples. Concentrations of GSH, uPA and PAI-1, and activity of GST were independent from histopathological and clinical prognostic factors.
Increased GSH concentration and GST activity found in primary malignant and metastatic ovarian tumor samples were independent of histopathological and clinical prognostic factors, suggesting that they could be early markers for ovarian carcinomas.
To estimate the prognostic value of cathepsins B, H, L, D and stefins A and B in head and neck carcinoma, their concentrations in cytosols of primary tumours and adjacent normal tissue were measured ...(cathepsins B, D stefins A, B in 45, cathepsin L in 24 and cathepsin H in 21 patients). Median concentrations of cathepsins B, L, and D were significantly higher in tumour than in the adjacent normal tissue (B and D: p < 0.0001; L: p = 0.004); cathepsin H concentration was higher in normal tissue (p = 0.001). Concentrations of either stefin did not differ significantly between normal and tumour tissue. Concentrations of cathepsins B, H, L, and D were higher in laryngeal than in non-laryngeal normal and tumour tissues. The difference was statistically significant for cathepsin B in tumour tissue (p = 0.045), and marginally significant in normal tissue (p = 0.07). Early tumours had lower concentrations of stefins A and B than locally advanced tumours (stefin A: p = 0.04; stefin B: p = 0.07). Disease-free and disease-specific survival rates were better in patients with concentrations of cathepsin L in tumour tissue below or equal to the cut-off values (p = 0.035; p = 0.05), whereas for cathepsin B the difference was established only for disease-free survival (p = 0.07). The opposite was true for stefin A (p = 0.0002; p = 0.002) and stefin B (p = 0.009; p = 0.003), and in disease-free survival also for cathepsin H (p = 0.055). The concentration of cathepsin D did not correlate with survival. Our data indicate that cathepsins B, H, L and stefins A and B might have prognostic value in head and neck carcinoma.
The aim of the study was to evaluate the prognostic significance of tumour and serum concentrations of urokinase-type plasminogen activator (uPA), its type 1 inhibitor (PAI-1) and cathepsin D (Cath ...D) in patients with squamous cell carcinoma of the head and neck (SCCHN).
Determinations of uPA and PAI-1 were made using enzyme-linked immunosorbent assays in tumour and serum samples of 47 and 32/47 patients, respectively. For the determination of tumour (94 patients) and serum (34/94 patients) Cath D concentrations, an immunoradiometric assay was used.
In an univariate survival analysis, the risk of disease recurrence and SCCHN-related death was significantly higher in the patients with high uPA (P = 0.046, P = 0.010) tumours, compared to those with low uPA tumours. In addition, the high serum levels of uPA correlated positively with the rate of relapse (P = 0.007), but not with the mortality rate (P = 0.200). There was no statistically significant difference between low and high PAI-1 groups, regarding either tumour or serum concentration of the inhibitor, and between low and high Cath D tumours. Low Cath D serum levels appeared to be related to longer disease-free interval (P = 0.055), but not to disease-specific survival (P = 0.120).
The tumour levels of uPA, as well as serum levels of uPA and Cath D could potentially predict the survival probability of patients with SCCHN. However, the strength of this association remains to be investigated on a larger and more homogeneous group of patients.
The effects of nine intra- and extracellular proteinases and six proteinase inhibitors on the repair of potentially lethal damage (PLDR) induced by gamma-rays in plateau-phase V79 cells were ...examined. It was demonstrated that these agents, which are intrinsic factors produced within mammalian cells, can modify PLDR activity. A stimulatory effect on PLDR was seen with calf liver neutral proteinase, and to a lesser extent, with inhibitor pepstatin A. Other proteinases which belong to serine, cysteine and aspartic superfamilies, as well as proteinase inhibitors, inhibited PLDR to different degrees. The effects of some of these agents, present during the PLDR period, on the rate of tritiated thymidine incorporation into the acid-insoluble cell fraction was also examined. They can modify the DNA synthesis of cells when subcultured from plateau phase for the assessment of colony-forming ability. There is no clear evidence that the effects observed are entirely attributed to the alteration of cellular proliferative processes. It seems more likely that many serine and cysteine proteinases and their inhibitors can adversely affect the PLDR process by modulating the activity of proteinase(s) and other enzymes involved more directly in PLDR because of interrelationships of the entire intracellular proteinase system.
