The novel exon 11 mutation of BRCA1 gene in a high-risk family.

Germline mutations in the BRCA1 and BRCA2 genes are required for the initiation of the development of hereditary forms of breast and ovarian cancer, which represent 10-15% of all cases. The course of the disease varies from case to case that can be due even to the possibility of multiple genetic changes including inactivation of other tumor suppressor genes--TP53 and APC genes or activation of oncogenes, especially K-ras oncogene. The combination of these changes results in an early expression of the broad variety of malignancies. The analyzed proband (II-5) comes from a high-risk family, in which various types of cancer were observed. The novel BRCA1 mutation in exon 11 (2057delCAGTGAAGAG) was detected by SSCP, HDA techniques and confirmed by automatic sequencing. The same deletion was observed in DNA sample of her first daughter (III-1), but DNA of her second one was without any mutational changes (III-2). Due to the occurrence of different types of cancer in this family, the incidental mutations in the APC; resp. TP53 tumor supressor genes and K-ras oncogene were searched as well. Any mutation was found after sequencing of SSCP interesting exons of these genes. The reasons for such strong malignant manifestation in this high risk family are discussed.

Mutation screening of the BRCA1 gene in Slovak patients.

Breast cancer is the most commonly observed malignancy in women of the western world. The family history is the strongest risk factor for the disease. Two major genes, BRCA1 and BRCA2 that are involved in the familial breast and ovarian cancer have been described. Germ-line mutations of the BRCA1 gene have been linked to 85% of all hereditary breast and ovarian cancers. We performed a mutation screening ofthe entire codingregion of the BRCA1 gene in 29 Slovak families suspected of having inherited predisposition to breast cancer. For the analysis we used a combination of a single strand conformation polymorphism (SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing. Genetic alterations were consistently indicated by SSCP and DHPLC and consequently confirmed by DNA sequencing as previously described pathogenic mutations. The patients with inherited BRCA1 mutations will undergo genetic counseling and cancer prevention health care program.

Activated lymphocytes from breast cancer patients express the characteristics of type 2 helper cells--a possible role for breast cancer-associated p43.

P43, a breast cancer-associated antigen, has been repeatedly described as an immunosuppressive factor. The objective of the present study was to investigate whether immune dysregulation induced by p43 affects the profile of cytokines secreted by mitogen-stimulated lymphocytes in breast cancer patients as compared with stimulated lymphocytes in women with benign tumors. The study consisted of 32 women undergoing surgical excision for a suspicious lesion in their breast. Histology revealed malignant breast disease in 20 patients and benign lesions in 10 patients. Lymphocytes isolated from peripheral blood were activated by Conconavalin A (Con A) with and without the addition of p43 and the concentrations of cytokines (IL-2, TNF-alpha, IFN-gamma, IL-4, IL-10 and IL-6) secreted into the culture medium were determined. Lymphocytes of patients with malignant breast disease stimulated with Con A secreted a significantly higher concentration of IL-10 compared with lymphocytes of patients with benign tumors. No significant differences were found between the two groups regarding the levels of IL-2, TNF-alpha, IFN-gamma and IL-4. Cytokine concentrations were analyzed according to the type 1/type 2 cytokine profile (IL-2, TNF and IFN-gamma and IL-4, IL-6 and IL-10, respectively). This analysis revealed no significant differences in IL-2, TNF or IFN-gamma between benign and malignant tumors. However, in the type 2 cytokines, lymphocytes from cancer patients secreted significantly higher levels of IL-4 (27.3 +/- 7.2 U/ml) and IL-10 (44.1 +/- 22.3 U/ml) than did the lymphocytes from patients with benign disease (21.4 +/- 7.3 and 1.8 +/- 0.3 U/ml, respectively). The addition of p43 to the culture medium significantly enhanced the levels of IL-4 secreted by lymphocytes in both groups of patients (malignant disease, from 27.3 +/- 9.2 to 40.7 +/- 6.3 U/ml; benign disease, from 21.4 +/- 7.3 to 28.4 +/- 2.1 U/ml). P43 antigen significantly enhanced the low levels of IL-10 in the benign lymphocytes (from 1.8 +/- 0.4 to 8.4 +/- 1.5 U/ml) while the high levels of IL-10 secreted by the PBL in patients with malignant tumors were not significantly increased (44.1 +/- 22.3 versus 50.1 +/- 12.6 U/ml). The study showed a difference in the immune response of lymphocytes between malignant and benign tumors. When the current results were analyzed according to the type of response, i.e. in terms of whether at least two cytokines of either type 1 or type 2 were elevated, a significant type 2 response was observed in the PBL of patients with malignant breast cancer (IL-10 and IL-4). These results may explain why antitumor response is impaired in patients with breast cancer.

Immunosuppression by breast cancer associated p43-effect of immunomodulators.

It has been previously shown that p43- a breast cancer associated antigen-has immunosuppressive properties. The present study was carried out in order to elucidate the pathomechanisms of immunosuppression in breast cancer patients influenced by the expression of p43. Lymphocytes were cultured from blood of 29 women with benign lesions in the breast as well as from 41 female patients with breast cancer. Lymphocyte stimulation was performed by addition of Concanavalin (Con A) in cultures with lymphocytes alone (CONLYM) or in lymphocytes incubated with p43 (CONAg). In other series immunomodulation was tried by addition of indomethacin (INDLYM, INDAg), levamisole (LEVLYM, LEVAg), or interleukin-2 (ILLYM, ILAG). In breast cancer patients, addition of p43 significantly inhibited the activation of lymphocyte proliferation by Con A compared to women with benign tumors. The addition of indomethacin or levamisole did not influence this inhibitory effect of p43 in breast cancer patients. Contrary to these observations, addition of IL-2 resulted in increased proliferation of lymphocytes from patients with benign as well as malignant tumors, which was inhibited after addition of p43. Analysis of the correlation of the immunosuppressive properties of p43 in correlation with prognostic factors for breast cancer showed evidence for a stronger activity of p43 in early stage tumors (i.e. smaller than 2 cm, lymph node negative, histologic grading GI), confirming previous observations of a higher expression of p43 in early stages of breast cancer.

Immunosuppressive activity of lymphocyte mitogenesis by breast cancer-associated p43.

Placental isoferritin (PLF), an acidic isoform of ferritin, and its unique superheavy chain of 43 kDa (p43) has been described to be synthesized by human breast cancer cells. Physiologically, p43 PLF produced by the placenta is involved in immune suppression of maternal lymphocytes aimed at fetal antigens. A study was carried out to elucidate a paradigm of p43 occurrence in breast cancer patients. Immunosuppression of cytotoxic CD8+ lymphocytes was measured via inhibition of blast transformation in concanavalin A (ConA) stimulated peripheral blood lymphocytes (PBL) using 3H-thymidine uptake in vitro. PBLs were cultivated from 29 women having benign lesions in the breast as well as from 41 patients with breast adenocarcinoma. In breast cancer patients addition of p43 significantly inhibited the activation of lymphocytes proliferation by ConA compared to women with benign tumors. The addition of indomethacin or levamisole did not influence this inhibitory effect of p43 in breast cancer patients. Presence of interleukin-2 in cultures was able to overcome the inhibitory effect of p43 on CD8+ lymphocytes proliferation from women having breast adenocarcinomas and to increase its value in patients with benign lesions.

Alterations of cell surface antigens induced by placental isoform of ferritin in human carcinoma cell lines.

AIM: the ability of the breast cancer-associated placental acid isoform of ferritin (p43-PLF) to modulate cell surface expression of HLA, CD59 (protectin) and CD66 antigen (adhesion antigen related to CEA) was examined. METHODS: the expression of these antigens in human breast carcinoma cell lines BT-20, T47D and MDA-MB-468 was determined with the aid of flow cytometry and monoclonal antibodies. RESULTS: PLF induced a transient up-regulation followed by a down regulation of cell surface protectin (CD59 antigen) on the cell surface of T47D and to a lesser extent, BT-20 human breast carcinoma cell lines. Furthermore, PLF down-regulated cell surface expression of CEA-related CD66 antigen on both these cell lines. No PLF-induced alterations of protectin, CD66 antigen and HLA class I antigen were found on the MDA-MB-468 breast cancer cell line. CONCLUSIONS: breast cancer-associated p43 induces alterations of the expression of cell surface molecules in breast cancer cells which could have an effect on the modulation of cancer cell adhesive interactions.

Downregulation of lymphocyte mitogenesis by breast cancer-associated p43.

Placental isoferritin-associated p43 has proven to induce immune suppression during pregnancy in order to avoid rejection of the fetus' alloantigens by maternal lymphocytes. It has been demonstrated previously that p43 is also synthesized by breast cancer cells and can also be found on the surface of a subpopulation of T cells in women with this disease. Therefore, it was the aim of the present study to investigate if breast cancer-associated p43 has immunosuppressive properties. In 40 women undergoing surgical excision of a suspicious lump in their breast, blood was withdrawn and lymphocytes were isolated. Lymphocyte cultures were incubated with p43 antigen and anti-p43 antibody (CM-H-9). In a second series, lymphocyte mitogenesis was activated by addition of concanavalin A (Con A), Con A + p43 and Con A + anti-p43, respectively. While lymphocytes of breast cancer patients (n = 21) and women with benign breast disease (n = 19) incubated with p43 as well as with anti-p43 antibody did not show any difference in terms of incorporation of [3H]thymidine, activation of lymphocytes by addition of Con A was significantly inhibited after addition of p43 antigen in breast cancer patients compared to women with benign breast disease (P = 0.0178). Analysis of prognostic factors for breast cancer showed that inhibition of lymphocyte mitogenesis was dependent on the degree of tumor differentiation and was significantly higher in well differentiated tumors (GI) compared with more dedifferentiated tumors (GIII). The present study shows that breast cancer-associated antigen p43 is able to induce immune suppression in breast cancer patients but not in women with benign breast disease.

Expression of p43 associated placental isoferritin (PLF) correlates inversely with cell growth in breast cancer cell lines MCF-7 and T47-D.

We have previously demonstrated that the expression of the recently described immunosuppressive antigen p43 in breast cancer patients correlates with early stages of the disease and a low degree of proliferation of the tumors. Attempts were made to evaluate the expression of p43 in two breast cancer cell lines (MCF-7 and T47-D) stimulated to proliferation by 17-beta estradiol and fetal bovine serum (FBS). p43 expression was determined by RIA technique using the new monoclonal antibody CM-H-9, the rate of proliferation was assessed by [3H]thymidine incorporation during 72 hours of incubation. Induction of proliferation by addition of 17-beta estradiol and FBS to serum-free tissue culture medium correlated with a decrease of p43 synthesis in both cell lines. The level of p43 expression in nonstimulated cells was low in comparison to that in cells cultivated routinely (15% FBS, no estrogen). However, the drop of p43 synthesis was significantly stronger in cell lines with estrogen stimulated proliferation. Our in vitro results confirmed previous clinical observations describing an inverse correlation between p43 synthesis and degree of proliferation and differentiation in breast cancer for the first time. However, the pathologic mechanisms leading to this phenomenon need to be elucidated.

Expression of p43 in breast cancer tissue, correlation with prognostic parameters.

Placental isoferritin (PLF) and its unique superheavy chain p43 have been recently described as being synthesized by breast cancer cell lines but not by normal breast epithelial cells. Since previous reports have demonstrated a correlation between the content of 'normal' ferritin in breast cancer tissue and the degree of differentiation and prognosis, we have determined p43 in the cytosol of 122 breast cancer samples by use of the new monoclonal antibody CM-H-9. The synthesis of p43 showed a significantly negative correlation with tumor size (P = 0.0001), histologic grading (P = 0.0038), nuclear pleomorphism (P = 0.0019), rate of mitosis (P = 0.0002), lymphocytic reaction (P = 0.0001) and a significantly direct correlation with estrogen receptor status (P = 0.0009). Although patients with a higher p43 content showed a trend for a better outcome (median follow-up: 61.4 months), an independent influence of the cytosolic p43 content on survival could not be confirmed by a multiple Cox model. Therefore it seems that p43's prognostic impact is linked to the highly significant correlation with features of differentiation although a statistical bias in the Cox model due to the limited number of patients must also be taken into account. On the other hand, the significant correlation of p43 expression with factors for good prognosis was striking and consistent and warrants further research of this tumor product.

Comparison of two defective hepatitis A virus strains adapted to cell cultures.

The replication of two defective hepatitis A virus strains in cell culture was examined. The w.t. HAS-15 strain growing in FRhK-4 cells produced infectious icosahedral virions 27 nm in size as well as round shaped particles with lipids attached to their surface. The morphogenesis of HAV was membrane-dependent and the detected particles were in various degree of maturation. The MBB 11/5 strain growing in PLC/PRF/5 cells produced mainly noninfectious empty procapsids without RNA genome. The translation of viral proteins was uninhibited in both strains. The reason for restricted replication competence of both strains seemed to be different. In HAS-15, highly efficient encapsidation of the progeny RNA positive-strand lowered the formation of replicative intermediate forms. In MBB 11/5, nearly exclusive empty procapsid production gave evidence for the failure of the VPg primer protein attachment to viral RNA. Changes in the efficacy of viral genome replication were a result of the adaptation of HAV to propagation in vitro.

Placental isoferritin associated p43 antigen correlates with features of high differentiation in breast cancer.

Placental isoferritin (PLF), an acidic isoform of ferritin, and its unique superheavy chain p43 have been recently described to be synthesized by breast cancer cell lines but not by normal breast epithelial cells. Since previous reports have demonstrated a correlation between the content of 'normal' ferritin in breast cancer tissue, degree of differentiation, and prognosis, we have tried to evaluate the correlation of p43 in the cytosol of 122 breast cancer samples with commonly applied prognostic factors and features of proliferation and differentiation. In parallel, we investigated the correlation of p43 expression in MCF-7 and T47-D breast cancer cell lines during proliferation induced by estradiol plus fetal calf serum (assessed by 3H-thymidine incorporation), compared to p43 expression in stationary non-stimulated cell cultures. The levels of p43 in breast cancer cytosols correlated significantly negatively with tumor size (p = 0.0001), histologic grading (p = 0.0038), nuclear pleomorphism (p = 0.0019), rate of mitosis (p = 0.0002), and lymphocytic reaction (p = 0.0001), and significantly directly with the estrogen receptor status (p = 0.0009). Although patients with a higher p43 content showed a trend for a better outcome (median follow-up: 61.4 months), an independent influence of the cytosolic p43 content on survival could not be confirmed by a multivariate Cox model. In accordance with the observed negative correlation of features of differentiation vs. p43 expression, induction of proliferation by estradiol plus FCS added to serum-free tissue culture medium correlated with a decrease of p43 synthesis in both cell lines. Expression of p43 in estrogen and FCS-absent media revealed also a decrease in relation to a low spontaneous proliferation. However, the drop of p43 synthesis was significantly stronger in cell lines with estrogen-stimulated proliferation. Our in vitro and cytosol results confirm recent clinical observations describing an inverse correlation of p43 synthesis with the degree of proliferation and differentiation in breast cancer. However, the pathologic mechanisms leading to this phenomenon as well as the negative correlation with lymphocytic infiltration are still unclear and need to be further elucidated.

Synergistic action of human fetal liver cells and growth hormone on stimulation and inhibition of myelopoiesis.

Human fetal hepatocytes from midtrimester fetuses were studied in their ability to support pluripotent hemopoietic progenitor cells (GEMM-CFU) and myeloid progenitor cells (GM-CFU). We compared the activity of fetal liver stroma to stimulate hemopoiesis of mixed and myeloid lineages without hormonal additional and by influence of human pituitary growth hormone (hGH). Colony-forming assays in a double-layer semisolid agar system were used. This study shows that addition of hGH to the fetal liver stroma has enhancing activity upon formation of myeloid colonies. It seems that this activity is not mediated through liver macrophages but by releasing other hepatocyte-derived factors, perhaps by somatomedins. A model of such regulation is proposed. Further, there were observed small, lymphocyte-like cells close to the germinal center of colonies. They are supposed to be the first maturated cells in myeloid colony cell population and to exert inhibitory role on pluripotent stem cell (PSC) self-renewal by direct cell-to-cell contact.

Release of granulocyte-macrophage colony-stimulating activity from human alveolar macrophages and its support of human myeloid blast cell proliferation.

The ability of human alveolar macrophages to support colony formation of precursor blast cells of the myeloid lineage was investigated. Myeloid blast cells were collected from patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and from the livers of fetuses aborted in the second trimester of gestation. It was found that the alveolar macrophages (AM) produced sufficient amount of colony-stimulating activity which culminated in the fourth week of in vitro cultivation. Conditioned media from AM supported the growth of multipotential blast cell colonies (GEMM-CFU) in AML and MDS, while in fetal hemopoiesis macrophage colonies preponderated. Preincubation with human interferon-alpha (IFN-alpha) can abrogate the production of the granulocyte-macrophage colony stimulating factor (GM-CSF) by AM. Media conditioned by AM were not able to compensate for cell-to-cell contact in long-term cultures of AML blast cells but CSFs released from AM in vivo can contribute to aggravation of the disease.

Radioimmunoscintigraphy of human malignant melanoma. I. Pharmacokinetics and localization of 125I-labeled monoclonal antibody RG-12 in thymectomized mice bearing renal melanoma xenotransplants.

The novel RG-12 monoclonal antibody (MoAb) recognizing a high-molecular-weight antigen of human melanoma cells was radioiodinated and its biodistribution and tumor imaging was determined in immunosuppressed mice bearing xenografted human malignant melanoma HMB-2. Control and tumor-bearing mice were injected with 6 micrograms of 125I-labeled RG-12 IgG (8.9 MBq 125I-IgG/animal). Clearance of the MoAb from plasma had a mean half life of 20.6 hours. At day 2 after injection, radiolabeled RG-12 IgG localized in the tumor was 1.43% of the injected dose bound per gram tissue (ID/g), whereas the localization in the healthy kidney was below 0.5%. Tumor to tissue ratio of MoAb accumulation was low for hepatic tissue (1.25) but high for spleen (3.30) and kidney (3.25), respectively. Scanning with a gamma camera localized tumor mass in the right kidney and implanted peritoneal metastases.

Bacterial expression of the p24 gag protein of the bovine leukaemia virus.

The possibility of expression of the gag gene of bovine leukaemia virus (BLV) in the bacterial system was investigated. The DNA fragment coding for the gag core 24 kDa protein of BLV was inserted into the pORF1 expression vector. The polypeptides expressed in E. coli were analysed by Western blotting. The bacterially synthesized antigens were detected by the serum of a BLV-infected cow and by mouse monoclonal antibodies against the native p24 gag protein.

Provirus integration of bovine leukemia virus into DNA of infected human myeloma cells.

Sixty clones of bovine leukemia virus-infected B-human myeloma ARH77 cells were isolated. The DNAs of all clones were examined for BLV provirus integration by Southern blotting analysis. Proviral sequences were found in DNAs of two clones. One of them (clone I B3) contained one proviral copy with a deletion of approximately 5.5 kb; the other one (clone I F9) carried three integrated proviruses. Viral proteins of 70,000, 42,000 and 35,000 M. W. were found in extracts of clone I F9. In clone I B3 only the 70,000 M.W. protein was detected.

Bovine leukemia provirus in the DNA of different infected host cells.

Bovine leukemia provirus is reported to be integrated in the DNA of different infected mammalian cells. We observed morphological transformation in BLV infected sheep fetal spleen, kidney, thymus and sternal cultures. The presence of BLV specific sequences in their genome was established after digestion with the restriction endonuclease EcoRI and hybridization with a BLV specific probe. Human myeloma ARH77 and myeloid K562 cells infected with BLV were virus productive as detected by a reverse transcriptase assay. The presence of proviral sequences was confirmed after Southern blotting analysis. Restriction digestion by SacI enzyme yielded a complete 8.9 kb BLV provirus in infected ARH77 cells and a smaller 7.5 kb BLV fragment in infected K562 cells.

Role of 3'-end of viral genome in tumor heteroinduction by the avian sarcoma virus.

Transformation defective virus was derived by restriction endonuclease cleavage from a clone of the avian sarcoma virus Schmidt-Ruppin strain, strongly oncogenic for rats. The transfection experiments of chicken cells by digested proviral DNA gave rise to transformation defective virus. The td virus was possible to recover in vivo in chickens. The tumors obtained after a long latent period contained the sarcoma virus which was able to transform chicken cells in vitro and to induce tumors in chickens. All viruses, parental, td- and recovered were of D subgroup specificity. The tumor induction experiments in rats have shown that the recovery of viral genome deletion in td mutant by cellular sequences was not enough to regain the oncogenicity for rats. The results stressed the importance of 3-end sequences of the virus genome, probably the sequences in C region for heteroinduction ability of the avian sarcoma virus.

Inhibition of colony-forming cells from bone marrow of leukemic patients by 3-oxauracil.

An in vitro system of colony forming cell inhibition was used for evaluation of 3-oxauracil (2,3-dihydro-1,3-6H-oxazine-2,6-dione) effectivity against bone marrow cells from 18 leukemic patients. The highest inhibitive activity of the 3-oxauracil was found in cases of acute lymphoblastic leukemia, comparing with bone marrow cells from patients with acute myeloblastic and chronic myeloid leukemia. According to the morphological characteristics of the formed colonies, lymphoblastic leukemia cells could be discerned from the myeloblastic ones in this system.