PS2 protein in breast carcinomas: cut-off value of estrogen-regulated expression.

This study includes 152 patients with histologically confirmed breast carcinoma. Steroid hormone receptors (SR), estrogen (ER) and progesteron (PR) receptors, and pS2 protein were assayed on the same cytosolic extract in accordance with the recommendation of EORTC. Our results showed menopausal- and histologic grade-related expression of pS2 protein. Unfavorable carcinoma subgroups, in relation to expression of pS2 protein were defined: postmenopausal carcinomas with histologic grade II, and pre-, as well as postmenopausal carcinomas with histologic grade III. There were overlappings of individual pS2 protein values between favorable and unfavorable carcinoma subgroups in relation to the expression of pS2 protein. Otherwise, no overlapping of pS2 protein values was obtained between ER-positive and ER-negative carcinomas within defined unfavorable menopausal - and histologic grade-related expression of pS2 protein subgroups. The highest pS2 protein level observed in ER-negative unfavorable subgroups (15 ng/mg) was considered as the cut-off value which defined estrogen-regulated expression of pS2 protein.

Influence of quercetin on B16 melanotic melanoma growth in C57BL/6 mice and on activity of some acid hydrolases in melanoma tissue.

Quercetin (QC) (5, 7, 3', 4' -tetra oxyflavonolol) is an ubiquitous flavonoid in many plants. The influence of QC on the growth of B16 melanotic melanoma in C57BL/6 mice and activity of some acid hydrolases in the tumor homogenates were investigated. Two series of experiments were carried out: In the first experimental group mice were inoculated s.c. with 10(6) of tumor cells (TC) suspended in 1 ml of saline. TC were obtained from the current serial passages. In the second series of experimental group mice were inoculated with melanoma cells preincubated 15 min. in different concentrations of QC. Mice of both series were divided into three subgroups. Mice of the first series were treated with QC i.p. every second day in a dose of 0.1 mg, 0.5 mg or 1.0 mg (total dose of 1.0 mg, 5.0 mg or 10.0 mg per mice). Animals of the second series did not obtain any treatment. After the nineteenth day of experiment the mice were killed, tumors excised and weighed. Tumor tissue pieces were homogenized for enzyme activity determination. Fragments of tumor tissue were taken for electron microscopy (EM) investigation. In mice injected i.p. with QC mean tumor weight was significantly higher than in control I. The mean tumor weight in the first experimental group was higher than in control from 170% to 196% and in the second experimental group from 69% to 147%. Enzymes activity was also higher in both experimental groups as compared to controls. Arylsulphatase activity in the first group was higher from 102% to 144% and in the second one - from 97% to 115% than in control I. Acid phosphatase activity was higher from 100% to 155% in the first experimental group and from 56% to 161% in the second one. Cathepsin D activity was greater from 133% to 333% and from 113% to 300%, respectively. EM studies revealed the presence of greater number of Golgi structures and primary lysosomes in experimental groups of tumors (mice treated with QC and mice with melanoma preincubated in QC). These results clearly indicate that QC significantly enhances melanotic melanoma growth and increases acid phosphatase and cathepsin D activity in these tumors. The mechanism of QC action on the melanotic melanoma is not fully understood and remains to be defined.

Seasonal variation in estrogen and progesterone receptor levels in breast cancer--a factor in data interpretation.

It is still widely accepted that evaluation of estrogen and progesterone receptors (ER, PR) in breast cancer tissue is one of the basic predictive indicators of hormonal therapy success. Evaluation of hormonal receptors is usually done once at the time of biopsy or surgery on the breast regardless of the effect of time. In a retrospective evaluation of 1301 medical records of breast cancer patients the authors wanted to identify any time period dependence in quantitative or qualitative hormonal status. Steroid receptors were measured in breast cancer tissues by cytosol-based ligand-binding technique. The results suggest a strong time period dependence in hormonal status. In premenopausal patients simultaneous evaluation of ER/PR revealed an increased frequency of both positive receptors in the autumn months, and a decrease in the spring months. In postmenopausal patients, both positive receptors were found more likely in the summer months and both negative in the late winter months. Our results suggest that one evaluation of hormonal receptor status does not necessarily provide an accurate estimate of the hormonal status in breast cancer patients.

Radiation-induced apoptosis and cell cycle progression in TP53-deficient human leukemia cell line HL-60.

Human promyelocytic leukemia (HL-60) cells were irradiated with 0.5-100 Gy of gamma radiation and studied for 48 h post irradiation to determine the mode of death and progression of cells through the phases of the cell cycle. HL-60 cells are much more sensitive to radiation-induced loss of clonogenicity (D0 = 2.2 Gy) than to induction of apoptosis at 6 h (D0 for nonapoptotic cells = 32.6 Gy). After doses 20-50 Gy, the onset of massive apoptosis occurred and nonapoptotic cells were in G1/G0 phase of the cell cycle. In contrast, 6 h after irradiation with doses 2.5-10 Gy maximum cells were in S-phase and 16-24 h after irradiation were arrested in G2-phase. Maximum apoptosis occurred 48 h after irradiation with doses 3.5-10 Gy, and cells that died by necrosis were found in 9-44%.

Intensive cyclic chemotherapy with unprocessed whole blood support in advanced breast cancer.

The aim of our project was to compare the efficacy of mobilised whole blood versus cryopreserved PBPC (peripheral blood progenitor cells) obtained by leukapheresis in the support of hematopoietic recovery in cyclic intensive chemotherapy. Twenty-nine women with breast carcinoma were treated. The mean age was 46 years. In stage III were 23, in stage IV were 6. They received 6 cycles of epirubicin 150 mg/m2 and cyclophosphamide 1250 mg/m2. In the first cycle, 24 hours after chemotherapy, application of G-CSF 5 microg/kg/day was started, and discontinued when leukaphereses and whole blood collections were done. Leukapheresed progenitors were then divided into 3 aliquots, cryopreserved and reinfused after the 4th, 5th and 6th chemotherapy cycles. Mobilised whole blood was collected on day 14 of the 1st and 2nd cycles and reinfused 24 hours after chemotherapy. The occurrence of grade IV leukopenia was 1.82 times higher with whole blood support and grade IV thrombocytopenia 2.64 times higher than in cycles with cryopreserved PBPC support. This resulted from the fact that in one application the numbers of CD34+ cells and CFU-GM were nearly double in cryoconcentrates. The yields of CD34+ cells in 450 ml of whole blood were 1.8 x 10(6)/kg, which is not sufficient for optimal hemopoietic recovery.

Peripheral progenitor cells (PBPC) in supportive care after high-dose chemotherapy in breast cancer.

Hemopoietic growth factors (HGF) and leukapheresed peripheral progenitor cells (PBPC) are increasingly used for supportive care in high-dose chemotherapy (HDC) of solid tumors. Presently, therapeutic protocols with cyclic HDC plus PBPC support are successfuly used in breast cancer patients. Administration of PBPC significantly influences hemopoietic recovery in terms of shortening the pancytopenia period which reduces the risk of dangerous complications, especially the risk of infection. As a certain controversy exists about efficacy of this therapy, large randomized studies are conducted to find more accurate conclusions. In 1998 National Cancer Institute (NCI) gave top priority to four randomized studies of HDC with PBPC support. In recent years, rising yields of PBPC are obtained. The use of new combinations and dosages of hemopoietic growth factors leads to a significant increase of progenitor cells circulating in peripheral blood. Effective mobilization regimens combinations of chemotherapy and cytokines - enable to increase the numbers of circulating progenitors as much as 100-fold. Another aspect, how to minimize the risks is to reduce the transplant volume and so reduce the amount of cryoprotective agent DMSO (dimethyl sulfoxide) and hemolysed erythrocytes. This led to the idea to use only whole blood enriched for PBPC. At present it has been used also in our patients. The results show that enriched whole blood can be used as sufficient substitution for support in intensive cyclic chemotherapy in breast cancer patients.

Estimation of changes in tubulin induced with etoposide in human leukemia cells line K-562 by immunofluorescence microscope.

This study was undertaken to examine the influence of etoposide on the distribution of tubulin in cells of human leukemia cell line K-562 by using immunofluorescence method. The cells were cultured with 5 different doses of etoposide: 0.2, 2, 20, 200, 2000 microM/ml. Changes in tubulin were dependant on concentration of the drug compared to untreated control cells. The changes occurred in distribution of tubulin in cells K-562 after treating them with 200 and 2000 microM/ml etoposide. Cells treated with 200 microM/ml etoposide showed intense diffuse staining of the entire cells. At the surface of the cells there were also intensively stained structures extended outwards from the surface as lamellipodias. Cells cultured with 2000 microM/ml etoposide were much bigger from other cells exposed to lower doses of etoposide and control cells. Distribution of tubulin throughout these cells with diffuse intensive labeling of the cell periphery was seen. Very bright protrusions formed in lamellipodias at the surface of the cells were also observed.

Biological activity of some 4-anilinoquinazolines: cytotoxic, genotoxic and antiprotease effects, induction of necrosis and changes of actin cytoskeleton.

Fourteen substituted 4-anilinoquinazolines have been tested for cytotoxic effect and structure activity relationships. The most active derivatives were substituted by chlorine or bromine group in the aromatic ring, in the pyrimidine ring by morpholine group and in the aniline skeleton by nitro group in position 4 or 2. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)-quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline inhibited growth of tumor cell lines HeLa, B16 and L1210. Mutagenic data provided by Ames test showed, that the compounds 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline and 2-(morpholin-1-yl)- 4-(4'-bromoanilino)quinazoline did not exhibit the mutagenic effect, whereas the compounds 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino) quinazoline increased slightly the number of revertants of the strain TA 98 without metabolic activation. Concentration 26 micromol/L of 6-bromo-2-(morpholin-1-yl)-4-anilinoquinazoline induced necrosis of tumor cells B16. Concentration 5.2 micromol/l induced a significant increase of filamentous actin in the transformed HepG2 cells. Derivatives 6-bromo-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline, 6-bromo-2-morpholin-1-yl)-4-anilinoquinazoline, 2-(morpholin-1-yl)-4-(4'-bromoanilino)quinazoline and 6-chloro-2-(morpholin-1-yl)-4-(4'-nitroanilino)quinazoline exhibited antiprotease effect on plasmine. This results could be relevant for the anticancer properties of these compounds.

Signal-averaged electrocardiography in survivors of Hodgkin's disease treated with and without dexrazoxane.

Doxorubicin is one of the most effective anticancer drug, but its usefulness is limited by the risk of developing cardiomyopathy, cardiac dysfunction and ventricular arrhythmias. Dexrazoxane is used to protect against doxorubicin cardiotoxicity. It is uncertain whether the dexrazoxane-mediated cardioprotective effect will be reflected in electrophysiological properties of the heart. The aim of the present study was to evaluate the occurrence of frequency-domain signal-averaged electrocardiographic (SAECG) abnormalities of the QRS complex and the initial ST segment in patients treated with and without dexrazoxane. Thirty children and young adults 2 months - 15 years after completion of doxorubicin-containing therapy for Hodgkin's disease were evaluated with SAECG. Patients from group I (n = 13) received combined therapy with doxorubicin and dexrazoxane (DOX/DZX), patients from group II (n = 17) received doxorubicin without dexrazoxane (DOX). Using fast Fourier transformation within the QRS complex and the initial ST segment, area ratio (AR) values 40-100/0-40 Hz were calculated. Significant differences in these frequency parameters in the QRS complex between DOX/DZX group and DOX group (19.45+/-12.72 vs 46.18+/-43.06; p = 0.03) might indicate protective effect of dexrazoxane on electrophysiological myocardial properties.

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.

The role of STIR MRI sequence in the evaluation of the breast following conservative surgery and radiotherapy.

The purpose of the study was to define the value of fat suppressed STIR sequence in the MRI of the conserved breast. To our knowledge, this study is the first clinical evaluation of STIR sequence in post-therapy conditions. Forty patients with early (T1-2, N0-1) invasive breast cancer underwent conservative surgery and postoperative radiotherapy. Routine follow-up examinations, including physical examination and mammography were supplemented with breast MRI in all cases 6-166 months (mean 27.6) after initial treatments. Three patients had bilateral cancer. Including follow-up (9 patients) MRI examinations, altogether 53 MRIs were available for analysis. An 0.5 T MRI (Elscint, Haifa, Israel) was used with double breast coil. Axial T1 and T2 weighted spin echo, STIR and 3D gradient echo dynamic sequences were performed. Pre- and postcontrast slices underwent serial subtraction. Twenty-eight circumscribed lesions were identified. All were well visualised on STIR sequence, regardless of histologic nature of lesions. One low grade DCIS was not detected by any sequence. Differential diagnosis between benign and malignant lesions was not possible by STIR sequence alone. STIR sequence was found to be more sensitive in the detection of treatment related breast edema and fluid collection, than T2 SE (spin-echo) sequence. Even the patients who were not good candidates for subtracted contrast enhanced dynamic studies - because of motion artefacts - could have been examined with satisfactory results. STIR is a very sensitive sequence for depicting circumscribed lesions and post-therapy complications, but not suitable for differentiation. It is a useful tool in the follow-up of patients with conserved breast subjected to radiotherapy.

The degree of bone marrow infiltration in patients with hairy cell leukemia treated with splenectomy compatible with long-term hematological remission.

To determine the degree of bone marrow infiltration with hairy cells which is compatible with long-term hematological remission in patients treated with splenectomy, we have investigated 7 patients surviving in hematological remission 61 to 255 months (median 184 months) after splenectomy. As hematological remission has been considered absence of hairy cells (HCs) in the peripheral blood, normalization of peripheral blood cell counts (hemoglobin 120 g/l, white cell count 4.0 x 10(9)/l, absolute granulocyte count 1.5 x 10(9)/l, platelet count 100 x 10(9)/l) and absence of lymfadenopathy and any other activity of the disease. For detection of HCs a very sensitive method of immunostaining with monoclonal antibody DBA.44 in our own modification has been used. Low values of sIL-2R which is considered to be a non invasive marker of tumor burden and activity in HCL were in agreement with the opinion that the bone marrow was the only locality of tumor involvement in splenectomized patients. Infiltration up to 20% with HCs (range 4% to 20%, median 10%) was found to be compatible with long-term hematological remission and long-term overall survival. Patient (No 1) with 30% infiltration of bone marrow with HCs, still normal peripheral blood cell counts, but a very high level of sIL-2R represents extraordinary finding which has been discussed in details.

The importance of interstitial radiotherapy in the treatment of the base of tongue tumors: a retrospective analysis.

The authors have reviewed their experience with interstitial brachytherapy for the base of tongue cancer with the purpose of introducing treatment strategy and technique and presenting results. Between January 1993 and May 1999 twenty-one patients with primary squamous cell cancer of the base of tongue (T1-4N0-2) were treated by interstitial radiotherapy (RT). Seventeen patients with advanced stage cancer received brachytherapy (BT) boost after 60-66 Gy teletherapy and 4 patients with early stage (T1-2N0) were managed by sole BT after tumor excision and elective neck node dissection in case of positive or very narrow (< 5 mm) margin. High-dose rate (HDR) after-loading unit (Ir-192 source) was used with rigid needles or flexible plastic tubes. The treatment plan was performed by PLATO 3D BT planning system. The mean dose of boost BT or sole BT was 20 Gy (12-24 Gy) and 27 Gy (24-30 Gy), respectively. All treatments were delivered on consecutive days with a twice daily fractionation schedule, except the rigid needle technique (n = 4), where the dose was 12 Gy with a single fraction. After definitive RT of advanced stage disease, the rate of complete or partial remission was 65% (11/17) and 35% (6/17), respectively. At a mean follow-up time of 32 months the local tumor control for the entire patient population was 62% (13/ 21). Five patients (24%) died of local and/or regional failure and sixteen patients (76%) are alive (6 with local and/or regional disease and 10 without evidence of disease). All of the four sole BT treated patients belong to the latter group. The incidence of grade 2 or grade 3 mucositis was 48% and 52%, respectively. To achieve good local control with adequate doses, avoiding surgical morbidity and associated functional loss and to minimize late radiation sequelae, the combination of percutan and interstitial RT seems to be very advantageous in the treatment of the advanced tumor of the base of tongue. For patients with early stage (T1-2N0) cancer, sole postoperative BT of the tumor bed - by positive or very narrow margins - seems to be a feasible option. However, more patients and longer follow-up is required to define the value of sole BT.

The efficacy of radiotherapy for vertebral hemangiomas.

Vertebral hemangiomas are benign, slowly growing tumors sometimes causing local pain in the spine and/or neurologic disorders. The present paper includes 14 cases of painful vertebral hemangiomas treated by radiotherapy. All patients were irradiated using standard fractionation scheme with a total dose 20-30 Gy. One month after the treatment complete pain relief was noted in 36% of cases, five months later in 67% of cases, but in the remaining cases partial pain relief was noted. No correlation between treatment outcome and different biological and technical factors was found. No dose-response relationship was noted. The results suggest that anti-inflamatory effect of radiation plays the major role in this kind of treatment and that radiotherapy for vertebral hemangiomas is easy, short and highly effective analgetic treatment modality.

Study of antioxidant effect of apigenin, luteolin and quercetin by DNA protective method.

A DNA protective capacity of three flavonoids, apigenin (AP), luteolin (LU) and quercetin (QU) against free radicals generated by H202, resp. Fe2+ is reported. This effect corresponding with scavenging of free radicals or with chelating of iron was assayed at two concentrations of flavonoids studied (1 microM and 10 microM). The quantitative analysis has shown that LU possesses the highest DNA protective effect of flavonoids investigated in the presence of H2O2. On the other hand, in the presence of 10 microM Fe2+, AP exhibited the highest DNA protective effect at the concentration of 1 microM and the following order was reached at the stoichiometric concentrations (10 microM) of Fe2+. It is believed that this discrepancy is caused by the ability of LU and QU iron-complex formation as it was separately investigated using UV-VIS spectrometry.

Arylamine N-acetyltransferase activities in human breast cancer tissues.

N-Acetyltransferase activities were determined in tumor (12 malignant and 6 benign) and control (non-cancerous) breast tissues from 18 female patients. The activities of matched 12 malignant tumor and control tissue cytosols showed 6 rapid, 4 intermediate and 2 slow acetylators based on p-aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates. Compared to the activities of slow acetylators, the rapid acetylators exhibited mean apparent Vmax values about 5- and 50-fold greater for p-aminobenzoic acid and sulfamethazine, respectively. No correlation was observed between the blood and breast tissue N-acetyltransferase (NAT1 and NAT2) activities. When the mean apparent N-acetyltransferase activities of the malignant and benign breast tumor tissues were compared, the results showed an increased activity for both p-aminobenzoic acid (PABA) and sulfamethazine (SMZ) acetylation in the malignant tissues compared to benign ones, and also control tissues showed lower activities compared to tumor tissues. Moreover, the mean NAT2 activity was about 2-fold greater in the malignant tissues when compared to NAT1 activity.

Expression of cyclin A in human leukemia cell lines HL-60 and K-562 at the level of light and electron microscope by using immunocytochemical methods.

In this study subcellular localization of cyclin A in the human promyelocytic leukemia cell line HL-60 and the human erythroleukemic K-562 cell line was examined by immunocytochemical methods. Studies were based on light and electron microscope evaluations. Cyclin A at the level of light microscope was present in 48% of the cells in HL-60 cell line and in 40% of the cells in K-562 line. Streptavidin-gold method was used for localization of cyclin A at the ultrastructural level. There was expression of cyclin A in the nucleus and in the cytoplasm. In the nucleus gold particles were seen to be associated with the condensed chromatin of the both leukemia cell lines. In the cytoplasm cyclin A was concentrated at a low level and was associated with ribosomes. Controls of the leukemia cells incubated with normal mouse serum showed no labeling at the light and electron microscope level.

Evaluation of MMP-1, MMP-8, MMP-9 serum levels in patients with adrenal tumors prior to and after surgery.

The aim of the study is to evaluate MMP-1, MMP-8 and MMP-9 serum levels in patients with adrenal tumors prior to and after surgery. Metalloproteinase-1 (MMP-1), MMP-8 and MMP-9 serum levels were evaluated in 43 patients operated on at our clinic between 1997-1999. Forty-one (95.3%) patients underwent adrenalectomy. Two (4.7%) patients were disqualified from surgery due to infiltration of adjacent tissues. MMP-1, MMP-8 and MMP-9 serum levels were determined at the admission and in case of surgery again one month after the operation. ELISA assay (K&D) was applied. Tumor type was determined on the basis of clinical, hormonal and histopathological examination. The correlation between MMP levels and tumor sizes was also evaluated. Patients were divided into 6 groups. Group I included 11 patients with adrenocortical carcinoma (4 with Cushing's syndrome and 7 with incidentalomas); group II--6 patients with benign hormonally active adrenocortical adenoma (4 with Cushing's syndrome and 2 with Conn's syndrome); group III--patients with benign, hormonally inactive adenocortical adenoma; group IV--6 patients with benign, hormonally active phaeochromocytoma; group V--4 patients with hormonally inactive phaeochromocytoma; group VI--5 patients with hormonally inactive adrenal tumors of extraglandular origin (2 myolipomas, 2 fibrolipomas, 1 hammartoma). The control group comprised 10 healthy individuals. Increased MMP-8 and MMP-9 levels were noted in patients with benign and malignant adrenal tumors. No increase of MMP levels was found in patients with tumors of extraglandular origin. The increased MMP-8 and MMP-9 levels occurred most frequently in patients with adrenocortical and hormonally active adrenomedullar cancer, and most rarely in patients with hormonally active adrenocortical tumors. MMP-8 and MMP-9 serum levels did not significantly differ between patients with adrenocortical incidentaloma cancers and in patients with benign incidentalomas. MMP-8 and MMP-9 levels were not increased in patients with inoperable adrenocortical cancers. Serum MMP-1 levels were not increased in patients with benign and malignant adrenal tumors. After surgery, MMP-8 and MMP-9 levels decreased significantly in patients with adrenocortical cancers, whereas the decrease of these MMPs in patients with benign tumors, although noticeable, was not statistically significant. MMP-8 and MMP-9 levels decreased significantly in all patients with increased preoperative levels, although they remained higher than the maximum normal values only in few patients (in 7 and 2 patients, respectively). No correlation between the levels of evaluated MMPs and tumor sizes were found.

Location and incidence of chromosome and chromatid breaks in patients with Hodgkin's disease or testicular tumors.

In 90 patients aged 17 to 35 who suffered from Hodgkin's disease (HD) or had testicular tumors (TT), the location of chromosome and chromatid breaks on individual chromosome segments was reviewed using an adapted Funes-Cravioto scheme, in addition to examining the percentage of structural chromosomal aberrations. On the basis of an analysis of 1121 breaks in patients with HD or TT, the results were presented graphically as multiples of the expected number of breaks for the normal population. Before the beginning of treatment, the number of structural chromosomal aberrations (SCA) in patients with TT or HD was equal to that in a control group of subjects with malignant diseases. This, however, does not apply to the location of chromosome and chromatid breaks. In patients with HD, the dominant unstable sites are located on group A2 chromosomes, segments 2 and 5, and on group B chromosomes, segment 5. In patients with TT, the number of chromosome and chromatid breaks is also increased on group A2 chromosomes, segments 2 and 5, and in addition, also on group B chromosomes, segment 4.

Polymer conjugated bovine seminal ribonuclease inhibits growth of solid tumors and development of metastases in mice.

Bovine seminal ribonuclease (BS-RNase) exerts a potent cytotoxic activity when administered intratumorally (i.t.) to the nude mice bearing human tumors. The ineffective treatment with intravenous (i.v.) or intraperitoneal (i.p.) administration led us to the synthesis of polymeric conjugates with BS-RNase to prevent it from degradation in the blood vessel. Hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification and a PHPMA-BS-RNase conjugates were prepared. Classic conjugate (P-BS) with BS-RNase bound to the polymer by its oligopeptide site chains was prepared by aminolytic reaction of the polymer precursor bearing reactive ester groups situated in the side chains of polymer, while star-like conjugate (S-BS) was synthesized by the reaction of PHPMA containing end-chain reactive group with BS-RNase in aqueous buffer solution at pH 8. In contrast to the total ineffectiveness of free BS-RNase administered i.v. at a daily dose 10 mg/kg, application of P-BS and S-BS conjugates at doses 2 mg/kg and 0.5 mg/kg caused significant inhibition of the growth of human melanoma in nude mice. On the base of these results the effect of i.v. administered S-BS on the metastatic process and the survival of C57Bl/6 inbred mice inoculated with B16 melanoma cells was investigated. Sixty per cent of mice treated with S-BS (0.5 mg/kg/day) survived 100 days without metastatic foci when the experiment terminated. The average survival time of the treated groups was 75.5 days compared to 32.7 days in the control group. BS-RNase conjugated to water soluble polymers appears to be the first BS RNase preparation which exerts anticancer and antimetastatic activity following its intravenous administration.

Effects of short term treatment with pentagastrin, proglumide, tamoxifen given separately or together with 5-fluorouracil on the growth in the murine transplantable Colon 38 cancer.

It is well known that 5-fluorouracil (5-FU) is the most effective drug in the treatment of colon cancer, however the positive response is small, only about 20%. On the other hand, it has been postulated that the growth of colon cancer depends on many growth factors, such as gastrin and estrogens. The search for new substances increasing the antitumor effect of 5-FU has lasted for many years. The aim of the present study was to examine the effects of pentagastrin (PEN, syntetic gastrin analogue), proglumide (PRO, a blocker of gastrin receptor) and tamoxifen (TAM, a partial estrogen antagonist) given separately or together with 5-FU on proliferation, apoptosis, necrosis and proliferation/apoptosis (P/A) ratio in the murine transplantable Colon 38 cancer. The male mice were implanted with a suspension of Colon 38 cells. After 7 days, the animals were treated with PEN (250 microg/kg b.w., twice daily), PRO (100 mg/kg. b.w., twice daily), TAM (10 microg/animal) separately or together with 5-FU (60 mg/kg b.w., once) for 2 days. The incorporation of bromodeoxyuridine (BrdU) into cell nuclei was used as an index of cell proliferation (labeling index--LI). The in situ labeling of nuclear DNA fragmentation according to TUNEL method was considered as an apoptotic index (AI). It was found that 5-FU increased the apoptosis, unexpectedly increased the LI and decreased the P/A ratio when compared to control. Both PEN and PRO increased the apoptosis and in the case of PRO decreased P/A ratio when compared to control. TAM did not affect any of the examined parameters. All of the investigated substances modify the 5-FU action: PEN and PRO on AI and LI and TAM on AI and P/A ratio. Necrosis was observed in 3 tumors treated with PEN + 5-FU, in 2 tumors of PRO + 5-FU group and in 1 tumor of group with 5-FU and with PEN. Further studies are needed to elucidate if those modification of 5-FU action by the examined substances will be useful in the inhibition of the growth of Colon 38 cancer.

Further studies of blood levels of some tumor markers in the area polluted by polychlorinated biphenyls and control population.

The levels of beta2-microglobulin (beta2-m), alpha-fetoprotein (AFP) and thyroglobulin (TG) were measured in the serum of 245 employees of chemical factory formerly producing polychlorinated biphenyls (PCB) consisting of 54 males (age range 24-65 years, median 45) and 191 females (age range 20-69 years, median 45). The control population consisted of 636 adults from control areas of northwest and east Slovakia. The frequency of beta2-microglobulin levels lower than 1.6 microg/ml in 242 employees of chemical factory was 76.8% (186/242) which was three times higher (P<0.001) than 24.4% (155/635) in 636 controls. Still more remarkable difference was obtained when using the cut/off level of 1.2 microg/ml, the frequency of such values in the employees being 45.4% (110/242) vs. 4.4% (28/635) in the controls. In contrast, no difference in alpha-fetoprotein levels was observed between the employees and the controls, the respective frequency of these < 5.0 ng/ml being 87.6% (212/242) vs. 86.2% (389/451) and these < 10.0 ng/ml being 100.0% (242/242) vs. 97.8% (441/451). Similarly, the frequency of normal thyroglobulin levels < 50.0 ng/ml) did not differ, being 95.6% (174/182) in the employees and 87.9% (87/99) in the controls. Most of a total of 20 cases with thyroglobulin level > 50.0 ng/ml showed sonographicaly enlarged and multinodular thyroid with focal or diffuse hypoechogenicity, three of them showed solitary nodule with a diameter > 10 mm. Although the decreased levels of beta2-microglobulin might be somehow related to the modulation of immune system, more plausible explanation appears to be the possible impairment of renal tubules by PCB similar to that caused by heavy metals resulting in increased urinary excretion of beta2-microglobulin and decrease of its blood level.

Micronuclei frequencies in exfoliated nasal mucosa cells from pathology and anatomy laboratory workers exposed to formaldehyde.

Formaldehyde (FA) is a widely used industrial chemical. Sufficient evidence exists to consider FA as an animal carcinogen. A possible causal role for FA may be considered likely for cancer of the nasopharynx and the nasal cavities in humans. The frequency of micronuclei (MN) in cells of the nasal mucosa was evaluated for 23 individuals in pathology and anatomy laboratories exposed to FA. Twenty-five healthy subjects were selected from the university and hospital staff as a control group. The measured air concentrations of FA in the breathing zone of the laboratory workers were between 2 and 4 ppm. The mean +/- SD values of nasal mucosa MN (per 1000) frequency from exposed and controls were 1.01 +/- 0.62 and 0.61 +/- 0.27, respectively (p < 0.01). Effect of smoking, age, sex and duration of exposure on the genotoxicity parameters analyzed were also evaluated. Our data suggest that low level exposure to FA is associated with cytogenetic changes in epithelial cells of the nasal region and that nasal mucosa cells exposed through respiration is an important target of FA-induced genotoxic effects.

High dose rate intraluminal brachytherapy in the treatment of malignant airway obstructions.

Endobronchial brachytherapy has been increasingly used in an effort to improve local control and relieve symptoms of malignant airway obstructions. Results of the high dose rate (HDR) intraluminal brachytherapy in 67 patients with inoperable endobronchial tumor treated by combination of teletherapy and brachytherapy with curative (group A ) or palliative (group B) intent, patients with recurrent tumors after previous radiotherapy treated by endobronchial brachytherapy alone (group C), and patients treated by brachytherapy without teletherapy (group D) are presented. Symptomatic improvement was achieved in 66%, 74%, 64% and bronchoscopic response in 70%, 85%, 78% of patients in groups A, B and C, respectively. Median survival was 365, 242 and 884 days from diagnosis and 245, 151 and 153 days from the first brachytherapy application in groups A, B and C, respectively. In group D complete bronchoscopic response was achieved in 3 of 4 patients with early tumor and partial response in 6 of 7 patients with advanced disease. We observed 4 acute and 9 late complications. Brachytherapy is an effective palliative treatment of malignant airway stenosis, but the effect on survival is not apparent.

Staging in untreated patients with small cell lung cancer.

In order to describe the real biological behavior of the small-cell lung cancer we have analyzed survival rates of 66 patients with small-cell lung cancer who did not receive any specific anti cancer therapy. Also, objective of this study was to evaluate the staging system of the small-cell lung cancer. Untreated small-cell lung cancer patients with limited stage disease had statistically significant (p < 0.05) better survival rates in comparison to patients with extensive stage disease. T and N factor of the TNM classification did not influence the survival in untreated small-cell lung cancer patients. It appears that the TNM staging system is not predicting survival probabilities of untreated patients with small-cell lung cancer, while the two-stage system appeared very well based on survival probabilities of these patients.

Repair of oxidative DNA damage--an important factor reducing cancer risk. Minireview.

Oxygen free radicals formed during normal aerobic cellular metabolism generate a variety of DNA lesions including modified bases, abasic sites and single strand breaks with blocked 3' termini. If left unrepaired, these damages may contribute to a number of degenerative processes, including cancer and aging. In most organisms, the repair of oxidative DNA lesions is supposed to be handled by the base excision repair (BER) pathway. BER is a multistep process that involves the sequential activity of several proteins, many of them were isolated and functionally characterized using the simple prokaryotic and lower eukaryotic model systems, Escherichia coli and Saccharomyces cerevisiae, respectively. As the amino acid sequence of DNA repair proteins is often well conserved from bacteria to man, our understanding of BER in higher eukaryotes drives extensively from the microbial models, namely from the yeast S. cerevisiae. Thus, results obtained on a simple yeast model are a source of new information, which can be used as a paradigm for all eukaryotic cells.

Evaluation of p53 and bcl-2 oncoprotein expression in precancerous lesions of the oral cavity.

The oral cavity is continually exposed to various traumas due to the effect of thermal, mechanical and chemical stimuli, which when accompanied by inflammatory states may promote the growth of neoplastic changes. Numerous studies have revealed a correlation between the expression of p53 and Bcl-2 proteins and the progression of neoplastic disease. It cannot be excluded that these proteins act as biomarkers of a neoplastic transformation threatening in precancerous states (including leukoplakia) or the already existing neoplastic transformation (e.g. in oral squamous cell carcinoma). The aim of the study was to evaluate the expression of p53 and Bcl-2 proteins in the proliferating epithelium in relation to leukoplakia degree and with regard to the lesions accompanied and not accompanied by squamous cell carcinomas. Fifty-five cases of proliferating changes in the oral epithelium (leukoplakia) were investigated. Group I contained 20 leukoplakias not accompanied by oral squamous cell carcinomas. Groups II, III and IV included 35 cases of changes in the vicinity of carcinomas on the lower lip (group II), in the front 2/3 of the tongue (group III) and in the oral floor (group IV). Staining was performed according to the immunohistochemical method with the use of monoclonal antibodies against human p53 protein (DAKO No M7001) and Bcl-2 (DAKO No M0887). A higher expression of p53 protein (54%) was found in leukoplakia changes coexisting with squamous cell carcinomas, compared with the non-accompanied ones (p53--45%). The results indicate a correlation between epithelial dysplasia degree and p53 and Bcl-2 protein expression--severe dysplasia occurred with an increase in the expression of both proteins. Leukoplakias situated in the vicinity of squamous cell carcinomas showed higher expression of p53 and Bcl-2 compared with the non-accompanied alterations. A correlation was also revealed between the location and p53 and Bcl-2 protein expression degree in the non-accompanied changes; no such correlations were found in proliferating epithelial changes adjacent to neoplastic tumors.

Induction of heat shock protein 70 in drug-resistant cells by anticancer drugs and hyperthermia.

The altered constitutive and inducible levels of heat shock proteins 70 (Hsp70) in drug-resistant cells may influence the efficiency of combined hyperthermia and anticancer drug treatment. In the present study, the constitutive levels of Hsp70 and induction of these proteins by hyperthermia and two anticancer drugs (used for resistance development) were determined in cervical and laryngeal carcinoma cells. The levels of Hsp70 were quantified by Western blot. Constitutive levels of Hsp70 were similar in parental and drug-resistant cells suggesting that Hsp70 is not involved in drug-resistance. Hyperthermic treatment induced Hsp70 in all examined cell lines but with different kinetics between drug-resistant and parental cells. Following the treatment with anticancer drugs, Hsp70 was induced only in cisplatin-resistant laryngeal cells. Kinetics of Hsp70 induction (stress-type and cell-type specific) was different in drug-resistant cells as compared to parental cells. The observed alterations in Hsp70 induction in drug resistant and parental cells should be taken into account when combined treatments (i. e. hyperthermia and anticancer drugs) are planned.

G1 phase of the cell cycle control and lung cancer: biological and clinical implications. Minireview.

Neoplastic diseases, including lung cancer are characterized by an uncoordinated cell growth. Cellular proliferation follows an orderly progression through the cell cycle, which is governed by protein complexes composed of cyclins and cyclin-dependent kinases. These complexes exert their regulatory function by phosphorylation of key proteins involved in cell cycle transitions. Abnormalities of cyclins and cyclin-dependent kinases have been reported and proposed to be oncogenic events. It appears that the molecular networking of these proteins and complexes exerts an impact on two fundamental cell cycle regulators; p53 and pRb. Interactions between these two nuclear proteins are being delineated, implying potential links between p53 and Rb in cell cycle control, apoptosis, and tumor progression. Furthermore, the detection of alterations in p53 and Rb pathways appears to be of clinical significance.

Shifts in expression of immunological cell markers in relapsed acute leukemia.

The immunophenotypic features of leukemia blast cells were analyzed in a group of 156 patients with different immunological subtypes of acute leukemia, both lymphoblastic and myeloblastic. Of the 58 patients for whom immunologic studies were performed at relapse, 42 (72%) showed changes in the expression of immunologic markers. The minor shifts in B-ALL were observed most frequently and concerned of the loss of CD34 antigen in 17 cases and the loss of cALLA (CD10) in 7 cases of B-ALL at the first relapse. The acquisition of cell markers was not frequently observed, only in four cases could be seen. HLA-DR molecules remained relatively constant from diagnosis to relapse. In 2 from 3 T-ALL cases the loss of CD1 and CD2 markers, respectively, was noticed at relapse. CD5 and CD7 markers were relatively stable. In AML cases at relapse the acquisition of CD13 marker (in 4 from 7 cases) was often observed. It was interesting that comparing to the B-ALL cases, the loss of CD34 marker in AML cases was stray. In one case the acquisition of this antigen at relapse was actually observed. The major interlineage shift was detected in one case of B-ALL, that was newly diagnosed at relapse as AML M4 and presented different cytogenetic features. This case provides strong connection with the treatment, as more recently epipodophyllotoxins (vumon in our patient) have been linked to the development of secondary AML associated with a shorter latency period. The immunophenotypic changes frequently occur at relapse in all acute leukemia types. The shifts (loss or acquisition) in expression of individual markers at relapse are bound with the first diagnosis and may have a relationship to the treatment and are important for correct assessment of minimal residual disease.