Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function.

Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices. In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately after isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degrees C in 5% CO2-humidified atmosphere and observed by fluorescence microscopy. Parallel electron microscopy was performed to compare fluorescence data with general cell morphology. To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method. In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers. This study indicates that the 'doxorubicin-fluorescence test' may be considered a simple and rapid procedure for assessing hepatocyte functional condition. It may provide valuable and 'real time' guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.

Thyroglobulin regulates follicular function and heterogeneity by suppressing thyroid-specific gene expression.

Thyroglobulin (TG) is the primary synthetic product of the thyroid and the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and lysosomal degradation can each modulate thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (the TSHR), increases thyroid hormone levels by upregulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of the thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex (MHC) class I gene expression, which is also regulated by TTF-1 and Pax-8 in the thyroid, is simultaneously decreased; this maintains self tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. We now show that follicular TG, 27S > 19S > 12S, counter-regulates TSH-increased thyroid-specific gene transcription by suppressing the expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS and TSHR genes, but increases class I expression. TG action involves an apical membrane TG-binding protein; however, it acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the start of TTF-1 transcription. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS and/or TSHR gene expression. TG activity is not duplicated by thyroid hormones or iodide. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism which regulates follicular function, regulates thyroid hormone secretion, and contributes to follicular heterogeneity.

In vivo expression of thyroid transcription factor-1 RNA and its relation to thyroid function and follicular heterogeneity: identification of follicular thyroglobulin as a feedback suppressor of thyroid transcription factor-1 RNA levels and thyroglobulin synthesis.

We used in situ hybridization to evaluate thyroid transcription factor-1 (TTF-1) RNA expression in individual follicles and related this to thyroglobulin (Tg) synthesis in vivo, as estimated by immunohistochemical analysis. We studied the thyroids of Wistar rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels, but affects follicular function and Tg accumulation in the follicular lumen very differently. We show that TTF-1 RNA levels in vivo correlate directly with an increase in the cytoplasmic accumulation of Tg within the cells of individual follicles. Because TTF-1 increases Tg gene expression, RNA levels, and protein synthesis in thyroid cell cultures and because there is no correlation with TSH-increased Tg degradation within the follicular lumen, the increased cytoplasmic accumulation of Tg in vivo is interpreted to reflect TTF-1-increased Tg synthesis. Increases in serum TSH levels in the PTU or T4 treated animals did not always correlate with increases in this measure of increased Tg synthesis; and TSH levels did not always correlate with changes in TTF-1 RNA levels that would be expected to accompany increased Tg synthesis. As one possibility, this suggested there might be a hitherto unrecognized suppressor of TTF-1 RNA levels and TSH-induced Tg synthesis in individual follicles. The immunohistochemical data suggested that this suppressor might be follicular Tg itself. Supporting this possibility, we show that physiological concentrations of highly purified 19S follicular Tg decrease TTF-1 RNA levels in rat FRTL-5 thyroid cells and inhibit the action of TSH to increase Tg synthesis. We therefore suggest that follicular Tg is a feedback autoregulator of thyroid function that can counterregulate TSH actions on thyroid function in vivo and in thyroid cells in culture. We suggest this phenomenon contributes to follicular heterogeneity in vivo.

Thyroid transcription factor 1 is calcium modulated and coordinately regulates genes involved in calcium homeostasis in C cells.

Thyroid transcription factor 1 (TTF-1) was identified for its critical role in thyroid-specific gene expression; its level in the thyroid is regulated by thyrotropin-increased cyclic AMP levels. TTF-1 was subsequently found in lung tissue, where it regulates surfactant expression, and in certain neural tissues, where its function is unknown. Ligands or signals regulating TTF-1 levels in lung or neural tissue are unknown. We recently identified TTF-1 in rat parafollicular C cells and parathyroid cells. In this report, we show that TTF-1 is present in the parafollicular C cells of multiple species and that it interacts with specific elements on the 5'-flanking regions of the extracellular Ca2+-sensing receptor (CaSR), calmodulin, and calcitonin genes in C cells. When intracellular Ca2+ levels are increased or decreased in C cells, by the calcium ionophore A23187, by physiologic concentrations of the P2 purinergic receptor ligand ATP, or by changes in extracellular Ca2+ levels, the promoter activity, RNA levels, and binding of TTF-1 to these genes are, respectively, decreased or increased. The changes in TTF-1 inversely alter CaSR gene and calcitonin gene expression. We show, therefore, that TTF-1 is a Ca2+-modulated transcription factor that coordinately regulates the activity of genes critical for Ca2+ homeostasis by parafollicular C cells. We hypothesize that TTF-1 similarly coordinates Ca2+-dependent gene expression in all cells in which TTF-1 and the CaSR are expressed, i. e., parathyroid cells, neural cells in the anterior pituitary or hippocampus, and keratinocytes.

Autoregulation of thyroid-specific gene transcription by thyroglobulin.

Thyroglobulin (TG), the primary synthetic product of the thyroid, is the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and degradation control thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (TSHR), increases thyroid hormone levels by up-regulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of several thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex class I gene expression, which also is regulated by TTF-1 and Pax-8 in the thyroid, is decreased simultaneously. This helps maintain self-tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. In this report we show that follicular TG counter-regulates TSH-increased, thyroid-specific gene transcription by suppressing expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS, and TSHR genes, but increases class I expression. TG acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the 5' flanking region of TTF-1. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS, and/or TSHR gene expression. The inhibitory effect of TG on gene expression is not duplicated by thyroid hormones or iodide and may be mediated by a TG-binding protein on the apical membrane. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism that limits follicular function and contributes to follicular heterogeneity.

Regulation of major histocompatibility (MHC) class II human leukocyte antigen-DR alpha gene expression in thyrocytes by single strand binding protein-1, a transcription factor that also regulates thyrotropin receptor and MHC class I gene expression.

The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility class II gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of class II genes that are necessary for interferon-gamma (IFNgamma) to overcome the normally suppressed state of the gene and induce aberrant class II expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DR alpha class II gene expression, as is IFNgamma or the class II trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DR alpha promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNgamma reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNgamma-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous class II RNA levels to levels measured in cells treated with IFNgamma. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the class II 5'-flanking region is incubated with extracts from IFNgamma-treated FRTL-5 cells and that have been previously associated with IFNgamma-induced aberrant class II expression. These data suggest that SSBP-1 is involved in the action of IFNgamma to overcome the normally suppressed state of the class II gene; it functions together with CIITA, whose expression is independently increased by IFNgamma. The effect of SSBP-1 as a positive regulator of class II promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant class II gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant class II expression, but, rather, TSH or IFNgamma treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNgamma to increase class II gene expression despite the fact IFNgamma increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DR alpha S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on class II gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the class II 5'-flanking region. As SSBP-1 is modulated by IFNgamma and is involved in class I and TSH receptor as well as class II gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity.

Cell cycle synchronization of FRTL5 cells. A physiological model system.

We describe a "physiological" cell cycle synchronization model system. FRTL5 cells, TSH-dependent for proliferation, were starved from TSH. The cell cycle phases and the expression of markers associated to different cycle phases were evaluated. TSH starvation blocks proliferation without provoking death and induces virtually all the cells to accumulate in G0/G1 phase. TSH readdition allows 30% of these cells to enter the S phase. DNA topoisomerase II 170-kDa isoform is not expressed in G0/G1 synchronized cells while it is expressed in logarithmic growing cells. The 180-kDa isoform is not expressed in G0/G1 synchronized cells while it is expressed in 20% of logarithmic growing cells regardless of the cycle phase. c-myc mRNA is not expressed in G0/G1 synchronized cells while it is detectable upon TSH readdition. This system provides a tool for the analysis of events associated with the G0/G1 phase and the transition from G0/G1 to S phase.

In vitro cultured stromal cells from human tonsils display a distinct phenotype and induce B cell adhesion and proliferation.

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM-1), CD49a (VLA-1), CD49b (VLA-2), CD49c (VLA-3), CD49e (VLA-5), CD49f (VLA-6), CD29, CD51, CD44 and produce vinculin, beta-tubulin, alpha-actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up-regulated CD54 (ICAM-1) and induced HLA-DR and CD106 (VCAM-1). HTSC constitutively produce interleukin (IL)-6 which is enhanced upon activation with TNF-alpha. IL-8 and granulocyte/macrophage colony-stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL-1 alpha, leukemia inhibitory factor and IL-7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL-2-activated B lymphocytes cocultured in direct cell-cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non-lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.

Effect of marrow processing on hematopoietic cell recovery in bone marrow harvests: focus on filtration.

Bone marrow processing requires a first step of filtration to remove small clots, bone fragments, fat cells and fibrin followed by centrifugation to separate mononuclear cells (MNC). These procedures cause a significant loss of cells potentially including hematopoietic stem cells (HSC). We therefore analyzed the cell recovery and phenotype of various fractions (whole marrow; filtered marrow; MNC collected after centrifugation; bone marrow fragments trapped by filtration) of bone marrow harvests (BMH) from patients with different hematological malignancies undergoing autologous bone marrow transplantation. Analysis of 25 BMH showed that the mean percentage of WBC and MNC recovered after filtration was respectively 92.28 +/- 7.42% and 92.3 +/- 9.05% of the original BMH while after centrifugation the percentage was 20.23 +/- 6.47% and 75.7 +/- 12.81%. The percentage of cells present in the tissue fragments trapped in the filters obtained from five BMH was only 3.93 +/- 1.25% (WBC) and 5.65 +/- 2.2% (MNC) of those originally present in the harvest. Phenotypic analysis performed on the same samples showed that there is no selective loss of MNC or CD34+ cells in the filtration process. Our data indicate that processing of BMH, in particular filtration of tissue fragments, does not affect the recovery of HSC.

Effect of stromal cells from human lymph nodes on the growth of osteosarcoma cell lines.

We investigated whether stromal cells obtained from human tonsils could interact and modulate the proliferation of the osteosarcoma cell in order to determine why lymph node metastases usually have a low incidence and remain occult using routine examinations. The effects of the supernatant of resting or activated stromal cells were analysed on osteoblastic cell proliferation of three different cell lines (HOS, U2, OS, MG-63). Only the proliferation of MG-63 was significantly inhibited. The direct adhesion of stromal cells to the osteosarcoma cell lines caused a greater inhibition of the proliferation of all three lines tested.