Prognostic significance of cysteine proteinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck.

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 Ps 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.

Prognostic value of total cathepsin B in invasive ductal carcinoma of the breast.

Total cathepsin B (catB) was determined by ELISA in 62 specimens of invasive ductal carcinoma of the breast. It was measured in microgram/g of tumour protein (microgram/gtp). The median catB was 91 micrograms/gtp, not varying significantly with T-stage or with age. It was higher in lymph-node negative (143 micrograms/gtp) than in lymph-node positive patients (49 micrograms/gtp) (P = 0.0005), in grade 3 (132 micrograms/gtp) than in grade 1 and 2 tumours (72 micrograms/gtp) (P = 0.07) and in hormone receptor-negative (155 micrograms/gtp) than in hormone receptor-positive tumours (72 micrograms/gtp) (P = 0.025). The recurrence-free survival (RFS) at 54 months for patients with tumours with catB < or = 23 micrograms/gtp was 22% and for catB > 23 micrograms/gtp, 68% (P = 0.0004). CatB > 23 micrograms/gtp did not significantly influence the RFS. Multivariate analysis showed that lymph nodes involvement (P = 0.003) and catB (P = 0.007) were independent prognostic factors.

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in tissue and serum of head and neck squamous cell carcinoma patients.

The aim of this study was to determine urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) concentrations in tumour and adjacent normal tissue samples from 58 patients, and in serum samples from 40 of 58 patients with squamous cell carcinoma of the head and neck obtained at diagnosis and after completion of therapy. uPA and PAI-1 serum concentrations were also measured in 28 healthy volunteers who served as controls. Measurements were made using enzyme-linked immunosorbent assay (ELISA) techniques. For both uPA and PAI-1, significantly elevated concentrations were measured in tumour tissue as compared with normal tissue (uPA: 8.89 versus 0.41 ng/mg total protein (mgp), P < 0.0001; PAI-1: 23.9 versus 1.47 ng/mgp, P < 0.0001). A statistically significant difference in uPA concentrations was found between normal laryngeal and nonlaryngeal tissue (0.52 versus 0.3 ng/mgp, P = 0.008), and in PAI-1 concentrations between T1 + 2 and T3 + 4 stage of disease (17.32 versus 35.63 ng/mgp, P = 0.04). The uPA concentrations positively correlated with those of PAI-1 measured in both tumour (Rs = 0.62, P < 0.0001) and normal tissue (Rs = 0.30, P = 0.02). In serum samples, lower concentrations of PAI-1 were measured in the control group than in patients with cancer (412.0 versus 680.5 ng/ml serum (mls), P = 0.0006). The time of collection of the serum sample did not influence uPA and PAI-1 concentrations, and no association was observed between their concentrations and any clinical and histopathological prognostic factors tested. Our results indicate that both uPA and PAI-1 may play a specific role in the process of invasion and metastasis, and might also be of prognostic value in squamous cell carcinoma of the head and neck.

The influence of cathepsin B and leupeptin on potentially lethal damage repair in mammalian cells.

Cell response to irradiation depends on many micro-environmental and intracellular factors. It is known that proteinases control many physiological functions and are also involved in progression of the cell cycle. They also could be involved in cell response to irradiation. In this work the influence of cathepsin B, which is one of the important lysosomal proteinases, and one of its inhibitors, leupeptin, on the potentially lethal damage repair (PLDR) was studied. Chinese hamster V79 cells were irradiated with gamma rays in the plateau-phase of growth. Immediately after irradiation cathepsin B or leupeptin were added to the growth medium. Four hours later, a determined sufficient period of time for maximal PLDR, the cells were replated to assess survival and mutation induction. Mutation frequency was determined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus using resistance to 6-thioguanine (6-TG). Simultaneously, the activity of cysteine, aspartic and serine proteinases were determined at different postirradiation intervals. The results show that when plateau-phase cells were incubated with cathepsin B during the postirradiation interval strong inhibition of PLDR was observed, accompanied with a reduced number of 6-TG resistant mutants. If leupeptin was added, more modest inhibition of PLDR was observed, accompanied with only slight reduction in the mutation frequency. The addition of cathepsin B or leupeptin to irradiated cells modified the activities of intracellular proteinases. As the highest alterations in proteinase activities were observed at the time when maximum repair of DNA lesions occurred, the biological consequences could involve a series of sequential steps in intracellular proteinase activities.

Cathepsin D in tissue and serum of patients with squamous cell carcinoma of the head and neck.

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 (< or =60 versus >60 years, 28.8 versus 32.8 pmol/mgp, P = 0.045) and histopathological tumour grade (G1+2 versus G3, 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 (Rs = -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.

Effect of the multidrug resistance modulator valspodar on serum cortisol levels in rabbits.

PURPOSE: To contribute to a better understanding of the physiological role of P-glycoprotein (P-gp) in the adrenal gland, we initiated our studies in rabbits. The aim of our study was to explore the effect of the selective multidrug resistance (MDR) modulator PSC 833 (valspodar) on serum cortisol in rabbits. METHODS: Baseline and corticotropin-stimulated serum cortisol levels were measured before and after valspodar treatment in adult male rabbits. Seven rabbits were treated with 50 mg/kg per dose and seven, with 75 mg/kg per dose of valspodar subcutaneously. Serum cortisol levels were determined by radioimmunoassay adjusted for expected values. RESULTS: Serum cortisol levels (baseline as well as corticotropin-stimulated) increased after both valspodar treatment regimens. The increase was dose-dependent and was higher for the baseline than for the corticotropin-stimulated values. Serum valspodar levels exceeding 1000 ng/ml were achieved in all except one animal in each group. We hypothesize that the increased serum cortisol levels were due to increased adrenocorticotropic hormone (ACTH) secretion after valspodar treatment, but, unfortunately, we could not measure ACTH properly in rabbits by means of the commercially available kits. CONCLUSIONS: Our study indicates that P-gp is not involved in steroid hormone secretion in the adrenal gland. This is evident from observations that serum cortisol levels were found to have increased rather than decreased in rabbits treated with a P-gp blocker and that the treated animals appeared healthy and normal. Since P-gp was found to play an important role in protection against xenobiotics in some other organs, further studies to explore the protective role of P-gp in the adrenal gland are warranted.

Urokinase-type plasminogen activator, plasminogen activator inhibitor type 1 and cathepsin D: analysis of their prognostic significance in squamous cell carcinoma of the head and neck.

BACKGROUND: 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). PATIENTS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Drug-resistant human laryngeal carcinoma cells have increased levels of cathepsin B.

In our previous work we showed that the drug-resistance of cervical carcinoma, laryngeal carcinoma and glioblastoma cells may be accompanied by increased levels of tumor markers for invasion and metastasis (i.e. urokinase-type plasminogen activator, plasminogen activator inhibitor type 1, and/or cathepsin D). In the present study we examined the concentration of cathepsins B, L and H in three drug-resistant clones isolated from human laryngeal carcinoma (HEp2). The basal levels of cathepsins B, L and H were determined by enzyme linked immunoabsorbent assay (ELISA). Our results showed that all three clones had an increased level of cathepsin B (in two clones an almost 4-fold increase was determined). The level of cathepsin L was altered (increased) only in VK2 clone, while the levels of cathepsin H were similar in parental cells and drug-resistant clones. Thus, our results suggest that drug-resistance may be accompanied by an increased level of cathepsin B, i.e. tumor associated protease, involved in invasion and metastasis.

Drug resistant tumor cells have increased levels of tumor markers for invasion and metastasis.

The association between drug-resistance and three markers for invasive capacity: cathepsin D (Cath D), urokinase type plasminogen activator (uPA) and inhibitor of plasminogen activator type 1 (PAI-1) was examined in nine cervical and laryngeal carcinoma cell lines resistant to different cytostatics. The level of Cath D was measured by solid phase two-site immunoradiometric assay, while uPA and PAI-1 concentrations were determined by use of ELISA. All drug resistant cell lines had increased concentration of cathepsin D. uPA levels were similar in parental and drug resistant cervical carcinoma cells, but significantly higher in all examined drug resistant laryngeal carcinoma cells. In cervical carcinoma cells, PAI-1 concentrations were similar in parental and cisplatin resistant, but significantly higher in doxorubicin resistant cells. In laryngeal carcinoma cells, no increase in concentrations of PAI-1 was determined in the three from five resistant cell lines. There was no uPA in conditioned medium of parental or drug resistant cells. PAI-1 was detected in conditioned medium. Its levels were significantly increased in the medium of two cervical and three laryngeal drug resistant carcinoma cells. Thus, our results suggest that drug-resistance may be accompanied by increased levels of tumor associated proteases and/or its inhibitor.

Modification of potentially lethal damage repair by some intrinsic intra- and extracellular agents: I. Proteinases and proteinase inhibitors.

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.

Changed delivery of boron to tumours using electroporation for boron neutron capture therapy with BSH.

For effective boron neutron capture therapy (BNCT) it is important that a sufficient concentration of boron (10B) is present in the tumour during irradiation. This requirement represents a specific problem. The aim of this study was to test whether electroporation can be used as a non-specific drug delivery system to increase the delivery of sodium borocaptate-10B (BSH) into MCF7 (breast carcinoma) and B16F1 (melanoma) tumour cells in vitro and in B16F1 tumours in vivo. For the in vitro determination of 10B uptake, the cells were incubated in medium containing BSH and exposed to electric pulses. Boron levels were determined by inductively coupled plasma atomic emission spectrometry. In vivo, tumours were exposed to electric pulses 3 min after intravenous BSH injection. At different times after exposure the 10B concentration was determined in tumours and in blood. A difference in the 10B accumulation in the two cell lines was observed after continuous incubation of cells with BSH. No accumulation of 10B was observed in MCF7 cells, whereas in B16F1 cells, 10B accumulated well and reached a plateau within 30 min. Electroporation of these cells resulted in an accumulation of 10B into MCF7 cells up to the level of 10B in B16F1 cells. In vivo, the application of electric pulses increased and prolonged the entrapment of 10B (BSH) in the B16F1 melanoma tumours. A sufficient concentration of 10B was present in the tumour exposed to electric pulses for up to 24 h. Boron was quickly washed out from the blood and the level was below the concentrations in the tumours exposed to electric pulses at 2 h. The results of this study show that electroporation may provide a tool to increase boron concentration in the cells that have impaired transport of BSH through the plasma membrane. Furthermore, prolonged entrapment of BSH in tumours in vivo may, in addition to electroporation, be caused by the modifying effect of electric pulses on blood flow.

The role of proteinases, interferons and hormones in proliferative activities of nonmalignant and malignant cells.

Proliferative responses, in form of colony forming ability, of nonmalignant Chinese hamster fibroblasts (V79) and malignant human laryngeal carcinoma cells (HEp) were examined after treatment with proteinases, proteinase inhibitors, interferons or hormones, in G0-phase, early G1-phase and G1/S point of the cell cycle. These phases of the cell cycle are of particular importance from the point of control and regulation of the proliferative activity of cells, and the effects of proteinases and other agents examined in this study occurred predominantly at these points. Among the agents tested, cysteine proteinase inhibitor (CPI) and calf liver neutral proteinase acted differently on malignant cells as compared to nonmalignant cells: they inhibited the proliferative commitment of malignant HEp cells in early G1-phase, while stimulated colony formation of V79 cells at the same point of the cell cycle.

Proteolytic activity in liver cells from mouse, rat, Ehrlich ascites carcinoma bearing mouse and in Ehrlich ascites carcinoma cells.

The activity of intracellular proteinases from Ehrlich ascites carcinoma bearing mice were compared with that from liver cells of normal mice and rats. The activity of intracellular proteinases was measured in the supernatant of Ehrlich ascites carcinoma cells homogenate. The activity of intracellular proteinases in normal mouse and normal rat liver were different at pH 3.5, pH 6.0 and pH 7.5. The activity in liver cells from Ehlrich ascites carcinoma bearing mouse at pH 3.5 was not significantly changed from normal mouse or rat liver cells, however at pH 6.0 and pH 7.5 the activity in the affected liver significantly decreased. The proteolytic activity in the supernatant of Ehrlich ascites tumor cell homogenate was 0.110 E750 mmu/mgN at pH 7.5, 0.154 E750 mmu/mgN at pH 3.5. The proteolytic activity at pH 6.0 was not detected in any experiment.

Stress and radiation carcinogenesis in mice.

In the present experiment irritation consisting of a combination of an optic signal followed by a mild electroshock administered at regular intervals was started in 2 groups of animals at the age of 3 months. At 4 months of age, one of the irritated and one of the nonirritated groups were exposed to whole-body gamma irradiation at 20 daily doses of 0.5 Gy (50 rad(, 1.4 Gy/min (140 rad/min), while the other 2 groups were sham-irradiated. The animals were autopsied and the specimens were microscopically studied for the presence of malignant tumors. Malignant tumors involving particularly the testes and lungs and leukosis were found in 29% of males, whereas in females the tumor incidence with mammary, pulmonary and ovarian involvement and leukosis was 39%. The irradiation decreased the minimum latency time in the irritated males and both female groups. In males, the irritation lowered the cumulative prevalence of malignant tumors, a significant decrease being noted at the age of 15 months. In females, it was increased, with a significant rise observed to occur at the end of the experiment. The opposite effects of irritation on the radiation carcinogenesis in males and females can be attributed to the irradiation-induced specific alterations of the gonads in females and, in part, to a longer survival time observed in the irradiated females.

Cathepsin B and L and stefin A and B levels as serum tumor markers in squamous cell carcinoma of the head and neck.

Cysteine proteinases cathepsin (Cath) B and L and their endogenous inhibitors stefin (Stef) A and B concentrations were measured using a quantitative immunosorbent assay (ELISA; KRKA d.d., Novo mesto, Slovenia) in serum samples from 35 patients with primary and 7 patients with recurrent squamous cell carcinoma of the head and neck (SCCHN), obtained at diagnosis (Serum no.1) and after therapy (Serum no. 2), and compared to sera from 30 (Stef B, 90) healthy volunteers. A significantly higher Stef A (P = 0.005) and lower Stef B (P < 0.001) concentrations were measured in patients' Serum no.1 than in controls, and the levels of Caths B and L and Stef A were found to be significantly elevated in Serum no.1 as compared to Serum no. 2 (P = 0.045, P = 0.041 and P = 0.024, respectively). The time of Serum no.2 collection did not influence the concentration of either Caths or Stefs in these samples, and no correlation was observed with the established prognostic factors for any of the parameters studied. Patients with subsequently diagnosed recurrent disease had a significantly lower Cath L concentration than those without evidence of relapse during follow up (P = 0.05). The risk of disease recurrence and SCCHN-related death correlated significantly with low Cath L serum levels (P = 0.012, P = 0.006). The serum levels of Cath B, Stef A and Stef B did not influence significantly the probability of survival.

Plasminogen activator inhibitor type 2: potential prognostic factor for endometrial carcinomas.

The clinical determination of proteases which are involved in carcinogenesis, invasion and metastasis may contribute to the detection of the early stage of disease, and to the prognostic assessment of patients with the cancer. The aim of the present study was to determine the level of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1) and plasminogen activator inhibitor type 2 (PAI-2) in normal and malignant tissues of corpus uteri and to evaluate the possible correlation with clinical and histopathological prognostic factors. UPA, PA-I and PAI-2 were determined by the ELISA assay in tissue cytosol of matched pair samples from 27 patients with endometrial carcinoma. Results show that significantly higher levels of these proteins were found in malignant than in normal tissue samples (uPA: 1.266 versus 0.633 ng/mg protein, PAI-1:4.468 versus 1.958 ng/mg protein, and PAI-2:3.428 versus 0.483 ng/ml protein). The levels of uPA and PAI-1 did not correlate with clinical staging or pathohistological grading. However, in tumor tissues with clinical stages II and III, myometrial invasion > 50%, and lymphovascular invasion, increased levels of PAI-2 were determined. Our results indicate that components of the plasminogen activation cascade are up-regulated in endometrial cancer and suggest the role of PAI-2 in determining invasive potential of endometrial carcinomas.

Characterization of human breast adenocarcinoma SK-BR-3 cells resistant to doxorubicin.

Doxorubicin shows a wide spectrum of activities in solid tumors, especially against breast carcinoma. The aim of this study was to examine if doxorubicin, when given at lower concentrations than applied in clinic, may induce changes in treated cells. With this purpose we developed human breast adenocarcinoma SK-BR-3 cell line resistant to doxorubicin. The sensitivity of these cells to doxorubicin and to some other cytostatics used in cancer treatment was determined by colorimetric MTT assay. Some parameters which may be of importance as prognostic factors in treatment of breast cancer were analyzed as well. The expression of genes involved in mitotic signal pathway (EGF, TGF alpha, EGF-R, erbB-2, erbB-3, c-myc and c-H-ras) was determined immunocytochemically. The concentrations of cathepsins were determined using quantitative immunoreactive assays (cathepsins B and L) or immunoradiometric assay (cathepsin D). The results revealed that even low doses of doxorubicin can induce numerous changes in treated cells: they become resistant to doxorubicin, and cross-resistant to several other cytostatics. The expression of the above mentioned genes involved in mitotic signal transduction, as well as cathepsins D and L, was similar in both parental and doxorubicin treated cells.

Carcinogenesis after sublethal ionizing irradiation and regular avoidance irritation.

160 mice of the BALB/C strain of both sexes, aged 3 months, were divided into four equal groups out of which two were regularly irritated by a combination of an optical signal and electrical stroke. After one month of irritation one nonirritated and one irritated group were whole body irradiated with a single dose of 6.65 Gy (1.83 Gy/min), the other two groups were sham irradiated. The mice lived until their spontaneous death or one year after irradiation, respectively, when the rest of the animals were sacrificed. The appearance of malignant tumors was noted. Irradiation shortened the survival time while the irritation had an appeasing, compensatory effect, more expressed in the males than in the females. After irradiation the number and assortment of the tumors increased and the latent period essentially shortened. In the irritated animals the number and assortment of the malignant tumors were reduced and a tendency for lengthening of the latent time period was seen; these differences, however, were not statistically significant. In spite of some differences the response in both sexes to irradiation and irritation or their combination was similar.

The effect of intracellular proteinases on transformation of human lymphocytes.

The effect of two intracellular proteinases, calf liver chymotrypsin-like neutral proteinase and human brain cathepsin H, and of two proteinase inhibitors, leupeptin and endogenous cysteine proteinases inhibitor, was studied on unstimulated and mitogen-stimulated peripheral human blood lymphocytes. Classification of blood samples into four types, on the basis of their potential for spontaneous and mitogen-stimulated transformation, helped in analyzing the results. Within some of these types, at least, there appears certain uniformity of response to proteinase action. Lymphocytes most susceptible to proteinase action were those that were insensitive to phytohemagglutinin mitogenic stimulus. The maximal effect of proteinases or their inhibitors was obtained most often when these agents were added to lymphocyte cultures 24 hours before mitogen. Both proteinases, although of quite different specificities, gave very often results of similar trend on the same lymphocyte culture. Moreover, this was followed also by the two inhibitors, which showed not only inhibitory, but also potentiating power of action on lymphocyte transformation. The regulatory nature of the action of proteinases within lymphocytes and other cells is underlined.

Biochemical and biological characteristics of leucocyte proteinase inhibitors.

Pig leucocytes contain inhibitors of neutral and thiol proteinases. These proteins could be isolated from post-granule supernatant fraction as well as from nuclear extract using ion exchange chromatography, gel chromatography and affinity chromatography. Inhibitors differ in molecular weight, isoelectric point, immunologically and their inhibition ability against tested enzymes.