The differential ultrastructural localization of immunoglobulin heavy and light chains in human haematopoietic cell lines

The ultrastructural localization of immunoglobulin heavy and light chains has been investigated in nine haematopoietic cell lines, using a technique which involves the treatment of lightly prefixed cells with saponin to allow penetration of the antibody-peroxidase conjugate. The synthesis and secretion of immunoglobulin was also studied in these cell lines.
Immunoglobulin was found to be localized in the cisternae and on the membranes of the rough endoplasmic reticulum, perinuclear space and/or Golgi apparatus. In each case staining for heavy chains was weak or absent in the perinuclear space while staining for light chains was usually strong. Additionally in three cell lines immunoperoxidase staining indicated that heavy chains were absent from the Golgi apparatus despite the observed presence of light chains in the Golgi apparatus and the secretion of combined immunoglobulin into the supernatant. The results obtained suggest compartmentalization of the synthesis of light and heavy chains and indicate that the technique of immunoelectron microscopy may significantly contribute to an understanding of the mechanisms involved in immunoglobulin synthesis, intracellular transport and secretion.     

Chromatin remodeling in estrogen-dependent and independent human breast cancer cell lines

Chromatin restricts the accessibility of DNA to regulatory factors; its remodeling over the regulatory regions contributes to the control of gene expression. An increasing number of evidence links defects in chromatin remodeling machinery and cancer. Our aim is to elucidate the role of chromatin structure in the control of the expression of hormone-induced genes in breast cell lines estrogen-dependent or -independent for growth. Mammary tumor growth is controlled by steroid hormones via their nuclear receptor and by growth factors via tyrosine kinase receptors. 50 % of these tumors elude to hormonal control. This limits the anti-estrogen therapy. As a model, we have analyzed in several cell lines the chromatin organization of the regulatory regions of two genes, pS2 that is associated with a good prognostic, and cathepsin D (catD) that is a bad prognostic marker. The expression of the two genes is estrogen-regulated in estrogen-dependent cell line MCF7. In contrast in the hormone-independent cell line MDA MB 231, pS2 is not expressed and catD is constitutively expressed. Within the regulatory regions of pS2 gene, we have localized two regions that undergo a hormone-dependent change in chromatin structure in MCF7 cells but not in MDA MB 231. The lack of chromatin remodeling in MDA MB 231 cells is not due to the absence of expression of the estrogen receptor in the cell line. The expression of pS2 gene can be correlated with chromatin remodeling over the regulatory regions of pS2 gene. In contrast catD regulatory regions did not display hormone-dependent changes in chromatin structure, suggesting that hormone regulation takes place within regions with a constitutively open chromatin structure.     

Cytotoxic efficacy of bendamustine in human leukemia and breast cancer cell lines

PURPOSE: The purpose of this study was to characterise bendamustine's cytotoxic and apoptotic activity in a panel of leukemia and breast cancer cell lines in comparison to its clastogenicity in murine bone marrow. METHODS: The cytotoxic effect of bendamustine was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT)-dye reduction assay. Induction of apoptosis was evidenced by DNA gel electrophoresis, nuclear staining, Western blot poly-(adenosine diphosphate-ribose) polymerase (PARP) cleavage, and flow cytometry. As a measure of hematological toxicity, the formation of chromosomal aberrations was investigated in bone marrow cells isolated from mice treated with low non-toxic doses of bendamustine and lomustine. RESULTS: Bendamustine was preferably active against leukemic cells of lymphoid origin and was found to induce apoptosis in SKW-3 and BV-173 cells as shown by oligonucleosomal DNA and nuclear fragmentation, PARP cleavage, and formation of a sub-G1 fraction. Myeloid and breast carcinoma cell lines were resistant towards bendamustine with the exception of HL-60 cells which exhibit an intermediate sensitivity. Bendamustine was found to have a very low clastogenic effect as compared with equimolar doses of lomustine. CONCLUSION: Taken together, the mode of action of bendamustine includes induction of apoptosis. The specific spectrum of activity and the unexpectedly low clastogenicity support the hypothesis that bendamustine in not a typical alkylating agent but exerts an additional mode of action, possibly as a purine antimetabolite.     

Increased expression of ADAM family members in human breast cancer and breast cancer cell lines

Purpose ADAMs (A Disintegrin and Metalloprotease) are multifunctional, membrane-bound cell surface glycoproteins, which have numerous functions in cell growth, differentiation, and motility. We wished to investigate the expression of ADAM 9, 10, 12, 15, and in human breast cancer.Methods Expression of ADAMs was determined in breast cancer specimens and the corresponding non-neoplastic breast tissue from 24 patients, and in the MCF-7 and MDA-MB 453 breast cancer cell lines via quantitative RT-PCR and immunohistochemistry. The effects of anti-ADAM antibodies on cell proliferation were assessed by measuring DNA-synthesis.Results Breast cancer tissue samples showed increased mRNA expression of ADAM 9, 12, and 17, whereas ADAM 10 and 15 were not differently expressed. Protein expression was studied by immunohistochemistry. All ADAMs were expressed in MCF-7 and MDA-MB453 cell lines, with the highest expression levels being observed for ADAM 9, 12, and 17. Application of anti-ADAM 15 and anti-ADAM 17 antibodies significantly inhibited the proliferation of both MCF-7 and MDA-MB453 breast cancer cell lines. In contrast, the growth of MCF-7 cells appeared to be stimulated by the administration of anti-ADAM 12 antibody.Conclusion The results of this study suggest that ADAMs are differentially expressed in human breast cancer and are capable of modulating tumour cell growth.     

Specific glycosphingolipids mediate epithelial-to-mesenchymal transition of human and mouse epithelial cell lines

Epithelial-to-mesenchymal cell transition (EMT) is a basic process in embryonic development and cancer progression. The present study demonstrates involvement of glycosphingolipids (GSLs) in the EMT process by using normal murine mammary gland NMuMG, human normal bladder HCV29, and human mammary carcinoma MCF7 cells. Treatment of these cells with d-threo-1-(3′,4′-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), the glucosylceramide (GlcCer) synthase inhibitor, which depletes all GSLs derived from GlcCer, (i) down-regulated expression of a major epithelial cell marker, E-cadherin; (ii) up-regulated expression of mesenchymal cell markers vimentin, fibronectin, and N-cadherin; (iii) enhanced haptotactic cell motility; and (iv) converted epithelial to fibroblastic morphology. These changes also were induced in these cell lines with TGF-β, which is a well-documented EMT inducer. A close association between specific GSL changes and EMT processes induced by EtDO-P4 or TGF-β is indicated by the following findings: (i) The enhanced cell motility of EtDO-P4-treated cells was abrogated by exogenous addition of GM2 or Gg4, but not GM1 or GM3, in all 3 cell lines. (ii) TGF-β treatment caused changes in the GSL composition of cells. Notably, Gg4 or GM2 was depleted or reduced in NMuMG, and GM2 was reduced in HCV29. (iii) Exogenous addition of Gg4 inhibited TGF-β-induced changes of morphology, motility, and levels of epithelial and mesenchymal markers. These observations indicate that specific GSLs play key roles in defining phenotypes associated with EMT and its reverse process (i.e., mesenchymal-to-epithelial transition).     

Biotransformation of melatonin in human breast cancer cell lines: role of sulfotransferase 1A1

The biologically active melatonin metabolite, 6-hydroxymelatonin (6-OHMel), is conjugated to form 6-hydroxymelatonin sulfate (6-OHMelS). To elucidate the role of the sulfotransferase (SULT) enzyme 1A1, considerably expressed in normal and malignant human breast cells, we measured the formation of 6-OHMelS by ELISA in hormone-dependent MCF-7 and hormone-independent MDA-MB231 (MDA) breast cancer cell lines after stable transfection with SULT1A1. In parent MDA cells, low SULT1A1 mRNA expression was associated with moderate 6-OHMelS formation as determined after application (24 hr) of 0.1 microM 6-OHMel. As expected, overexpression of SULT1A1 in MDA cells resulted in a 2.9- and 110-fold increase in 6-OHMelS in the cytosol and cellular supernatant respectively. Furthermore, 6.3- and 115-fold increases were observed after 0.5 microM, and 12.6- and 101-fold increases after 1 microM 6-OHMel respectively. In MCF-7 cells, because of high basal SULT1A1 expression, only two- to threefold increases in 6-OHMelS were observed after transfection with the enzyme. In total, 866 and 539 pmol/mg protein 6-OHMelS were formed from 1 microM 6-OHMel in SULT1A1 overexpressing MDA and MCF-7 cells, respectively, whereas application of 1 microM melatonin produced only <1% of 6-OHMelS. Possible interactions with the SULT1A1 substrate tamoxifen (tam), an anti-estrogen applied in the therapy of breast cancer, were also studied. A concentration of 1 microM tam increased 6-OHMelS formation by approximately threefold in the presence of 1 microM melatonin or 1 microM 6-OHMel respectively. However, no alterations were detected after application of 1 microM 4-hydroxy-tamoxifen. In summary, we demonstrate the importance of SULT1A1 for the biotransformation of 6-OHMel in human breast cancer cells. Our data further suggest that tam can modulate melatonin biotransformation.     

ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines

Corticotropin (ACTH) binds to specific receptors in the adrenal cortex and thereby regulates glucocorticoid and mineralocorticoid production. The number of ACTH binding sites on adrenocortical cells is increased by exposure of cells to activators of the cAMP pathway. The mechanism responsible for the increase in ACTH binding sites is not known. We therefore studied the levels of ACTH-R mRNA in mouse Y-l and human NCI-H295 (H295) adrenocortical carcinoma cell lines. ACTH induced an increase in mouse ACTH-R mRNA in Y-l cells that was time and dose dependent, increasing 6-fold over basal levels following exposure to 10⁻⁸ M ACTH for 19–24 h. The amount of human ACTH-R mRNA in H295 cells increased 2–4-fold following a 24 h exposure to 10⁻⁸ M ACTH, 1 mM dbcAMP, or 10⁻⁵ M Forskolin. Treatment of H295 cells with angiotensin II (A-II) was found to dramatically increase the level of ACTH-R mRNA. These data indicate that regulation of ACTH-R mRNA levels is at least one mechanism by which ACTH and A-II elevate the number of ACTH binding sites in the adrenocortical cell.     

The relationship between growth and androstenedione metabolism in four cell lines of human breast carcinoma cells in culture

The conversion of androstenedione (A) to estrogens, testosterone (T) and 5 alpha-reduced metabolites was studied in different phases of cell growth in 4 lines of cultured human breast carcinoma cells. Aromatase activity was 10-fold greater in MD and DM than in MCF7 cells and was undetectable in ZR75 cells. Estrogen formation in MD and DM lines increased during the phase of exponential growth and decreased to 20% of maximum during confluence. 5 alpha-Reductase activity was determined by the formation of 5 alpha-androstane-3,17-dione (5 alpha-A-dione) and androsterone (AND), and was 5-fold greater in ZR75 cells than MD cells and 2-fold greater than in MCF7 cells. This activity was relatively constant during exponential growth and decreased during confluence. T accumulation was inversely related to 5 alpha-reductase activity. The MCF7 and ZR75 cells which contain estrogen receptors had the highest levels of 5 alpha-reductase activity while the MD line which lacks estrogen receptors had the lowest 5 alpha-reductase activity. The assessment of aromatase and 5 alpha-reductase activity in addition to estrogen and progesterone receptors may be helpful in predicting hormone sensitivity in human breast tumours.     

Calcium-binding proteins in human carcinoma cell lines.

Elevated levels of Ca2+-binding proteins are reported in transformed cells and thought to be involved in their uncontrolled proliferation and often increased motility. Therefore, three cell lines [LICR(Lond)-HN 1, -HN 2, and -HN 6] derived from human carcinomas displaying various degrees of locomotive activity were investigated for the presence of parvalbumin and related Ca2+-binding proteins. By applying different immunohistochemical methods in conjunction with a monospecific anti-parvalbumin antiserum, an intense staining was seen in cells displaying translocative motility. Often in these cells, an association with filamentous structures located in the nuclear region was observed. Unique Ca2+-binding proteins, absent from comparable normal tissue, were found in the malignant cell lines when analyzed by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatography. From these tumor cells a protein (Mr, 12,000; pI, 4.8) was isolated that crossreacted with antiparvalbumin antiserum. Peptide maps of this protein revealed a further structural homology to parvalbumin (Mr, 12,000; pI, 4.9). In analogy to muscle, where there is evidence for a regulatory role of parvalbumin in the contraction-relaxation cycle, we speculate that this protein is tumor-associated and connected to the motile behavior of carcinoma cells.     

Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain.

Prodynorphin-derived peptides [dynorphin A (Dyn A)-(1-17), Dyn A-(1-8), Dyn B, alpha-neo-endorphin, and beta-neo-endorphin] and proenkephalin-derived peptides [[Leu]enkephalin [( Leu]Enk) and [Met]enkephalin-Arg6-Gly7-Leu8 [( Met]Enk-Arg-Gly-Leu]) in selected brain areas of the rat were measured by specific radioimmunoassays. We report here that different regions of rat brain contain strikingly different proportions of the prodynorphin and proenkephalin-derived peptides. There is a molar excess of alpha-neo-endorphin-derived peptides over Dyn B and Dyn A-derived peptides in many brain areas. [Leu]Enk concentrations exceed those of [Met]Enk-Arg-Gly-Leu in certain brain areas such as the substantia nigra, dentate gyrus, globus pallidus, and median eminence (areas rich in dynorphin-related peptides). These results indicated that (i) there is differential processing of prodynorphin in different brain regions and (ii) [Leu]Enk may be derived from Dyn A or Dyn B (or both). In certain brain regions [Leu]Enk may derive from two separate precursors (prodynorphin and proenkephalin) in two distinct neuronal systems.     

Changes in microvessel endothelial cell gene expression in an in vitro human breast tumour endothelial cell model

Selective targeting of tumour-endothelium has been proposed as a means of therapy. The successful exploitation of this approach will rely on the identification of suitable targets expressed specifically on the tumour-associated endothelium. In an attempt to identify novel tumour-endothelium associated targets we have used differential mRNA display to identify genes up-regulated in an in vitro breast tumour-endothelial cell culture model. Confluent monolayers of human mammary microvessel endothelial cells (HuMMEC) were incubated for 5 days with MDA-MB-231 breast adenocarcinoma cell-conditioned medium (TCM). mRNAs isolated from TCM-treated and control cells were amplified using 104 combinations of four 3^′ anchored T₁₂VN primers and 26 ‘random’ 10mers by RT-PCR and the products examined on DNA sequencing gels. Seventy-four sequences were cloned and the differential expression of five genes was confirmed using dot-blots. These were identified as procollagen type-IV, Tie-2/Tek receptor tyrosine kinase, NADH dehydrogenase subunit-6, and ferritin heavy-chain, which were up-regulated, and insulin-like growth factor binding protein-5, which was down-regulated. Increased endothelial expression of basement membrane proteins and tyrosine kinase receptors is known to occur during angiogenesis. Our data support the use of this model for further in vitro investigation of tumour angiogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production and pre-clinical significance of haematopoietic peptide growth factors (HPGF) in human non-seminomatous germ cell tumour cell lines

Peptide growth factors involved in the regulation of haematopoiesis (HPGF), for example granulocyte-colony-stimulating factor (G-CSF) and granulocyte/macrophage-colony-stimulating factor (GM-CSF), are of clinical importance in the treatment of testicular germ cell tumour (GCT) patients with modern chemotherapy regimens since they ameliorate chemotherapy-induced neutropenia. Aberrant expression of and/or response to HPGF has been reported in several solid tumour types although no data are available on GCT with the exception of those on stem cell factor (SCF). The aims of this pre-clinical study were twofold: (1) to screen a panel of human non-seminomatous (NS)GCT for the production of HPGF and (2) to test the effects of G-CSF or SCF on the growth of NSGCT cell lines in vitro, and on the growth kinetics of two human NSGCT xenograft models. HPGF concentrations in cell culture supernatant from 11 NSGCT cell lines growing under routine culture conditions were measured by enzyme-linked immunosorbent assay. The growth kinetics of cell lines was quantified in vitro using the sulphorhodamine B assay. The growth kinetics of nude mouse NSGCT xenografts was followed by measuring tumour volumes every 2–3 days over days 1–30, following daily subcutaneous injection of nude mice (days 1–14). The cell lines produced G-CSF (1/11 cell lines), GM-CSF (2/11), SCF (2/11), M-CSF (6/11), and interleukin-6 (9/11). Growth stimulation of cell line H12.1 by SCF was observed in vitro, but no statistically significant differences in NSGCT xenograft tumour volume (V _T) or relative V _T (rV _T) in treated groups were observed on days 14 or 29 compared to the control. The change in rV _T of H12.1 xenografts treated with G-CSF alone compared to control (rV _T/rV _T,c) was 0.96 on day 29. The values for rV _T/rV _T,c for H12.1 xenografts treated with G-CSF in combination with low- or high-dose SCF were, respectively, 1.67 or 1.7 compared to 1.19 for SCF-treated mice. The results are in agreement with clinical data to date where no observations have been reported of stimulation or inhibition of tumours in patients receiving treatment with G-CSF. Before any clinical trials are initiated in GCT patients treated with G-CSF in combination with SCF, further pre-clinical experiments with this tumour type are recommended to investigate this phenomenon further in a greater number of NSGCT cell lines in vitro and in vivo and with a wider range of SCF/G-CSF schedules. The potential relevance of secretion of HPGF in NSGCT cell lines in vitro to the pathobiology of GCT in patients is also a subject of interest for future research. Received: 12 May 1997 / Accepted: 19 April 1998     

Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant interferon-beta 2.

Recombinant human interferon-beta 2 was produced in Escherichia coli by direct expression of cDNA encoding the mature protein sequence. At concentrations that stimulate DNA synthesis and growth in B-cell hybridomas and plasmacytomas, the cytokine was found to exert a strong inhibition on the growth of a number of carcinoma and leukemia/lymphoma cell lines. This antigrowth effect was observed in clonogenic assays and by measurements of cell number and thymidine incorporation in growing cultures. The effect was blocked by antibodies to a synthetic peptide from the N terminus of the molecule. Normal diploid fibroblasts were inhibited at concentrations higher than those needed for breast carcinoma cells.     

Adrenal opioid proteins of 8600 and 12,600 daltons: intermediates in proenkephalin processing.

[Met]Enkephalin-containing proteins of 8600 and 12,600 daltons have been isolated from acid extracts of bovine adrenal medulla and purified to homogeneity, and their sequences have been determined by a combination of automated Edman degradation, tryptic mapping, and enzymatic time-course hydrolysis. The 8600-dalton protein contains one copy of the [Met]enkephalin sequence at the COOH terminus and the 12,600-dalton protein contains three copies of [Met]enkephalin, of which two are internal and the third is at the COOH terminus. They possess identical NH2-terminal amino acid sequences, suggesting that the 8600-dalton protein is derived from the 12,600-dalton protein by intracellular proteolytic processing. This is supported by results from tryptic maps of both proteins. Furthermore, chemical analysis of the tryptic peptides obtained from the 12,600-dalton protein indicates that it also contains the amino acid sequence that corresponds to a previously characterized enkephalin-containing polypeptide of 3800 daltons (peptide F) [Jones et al. (1980) Arch. Biochem. Biophys. 204, 392-395]. All three polypeptides appear to be intermediates in posttranslational processing of a still larger polyenkephalin precursor molecule, proenkephalin, and part of a biosynthetic pathway leading to smaller enkephalin-containing polypeptides and free enkephalins.     

Two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin.

Two enkephalin-containing polypeptides of 3600 and 4900 daltons have been isolated from extracts of bovine adrenal medulla, purified to homogeneity, and analyzed by a combination of automated Edman degradation and enzymatic time course hydrolysis. The 4900-dalton polypeptide contains two copies of enkephalin, one an internal [Met]enkephalin sequence, the other a [Leu]enkephalin sequence at the carboxyl terminus. Sequence analysis of the 3600-dalton polypeptide has not been completed, but the polypeptide has been shown to contain a single [Met]enkephalin sequence followed by an -Arg-Phe linkage that forms the carboxyl terminus of the molecule. On the basis of these and other findings, we propose that the above enkephalin-containing polypeptides are intermediates in the biosynthesis of the enkephalins and that they are generated by posttranslational processing from a large multivalent enkephalin precursor molecule, proenkephalin. The term "multivalent" is used to indicate a polypeptide with many identical functional sequences.     

Radioimmunoassay for human insulin-like growth factor-I receptor: applicability to breast carcinoma specimens and cell lines.

A radioimmunoassay for the human insulin-like growth factor-I (IGF-I) receptor was developed using a rabbit polyclonal antibody to the human IGF-I receptor and a highly purified IGF-I receptor. The purified receptor was radiolabeled with 125I-Bolton-Hunter reagent. Over 18% of the radiolabeled receptor was immunoprecipitated with the polyclonal antireceptor antibody. Purified IGF-I receptor concentrations as low as 5 ng/0.5 mL inhibited the radiolabeled IGF-I receptor binding. Purified insulin receptor weakly inhibited this binding, while the ligand IGF-I did not show inhibition. The radioimmunoassay was applicable to the measurements of IGF-I receptors in the Triton X-100 extracts of various tissues and cells. Breast cancer tissues and cells showed detectable IGF-I receptors, which correlated with IGF-I ligand binding. Receptor content was measurable in placenta and IM-9 cells, but receptor content was not measurable in liver and muscle extracts.