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PubMed2001

Differential expression of Hox A5 in human colon cancer cell differentiation: a quantitative study using real-time RT-PCR.

Fifteen different homeobox genes were identified from normal colon mucosa, untreated COLO 205 and herbimycin A treated COLO 205 cells in a degenerate primer RT-PCR screen. Several of the homeobox genes, including Cdx-1, Cdx-2, Pdx-1 and Hox A5, showed a trend toward differential expression in normal colon mucosa, and undifferentiated COLO 205 cells. Hox A5 was recently shown to suppress growth and induce p53-dependent apoptosis. To determine if Hox A5 was differentially expressed in differentiation of colon epithelial cells, we quantified Hox A5 expression by real-time quantitative RT-PCR. Expression of Hox A5 was 5.3- and 4.8-fold higher in normal colon mucosa compared to COLO 205 and HT-29 cells, respectively, suggesting that Hox A5 expression was higher in differentiated compared to undifferentiated colon epithelial cells. To avoid the complexity of tissue specimens and the influence of individual variation in Hox A5 expression, the effect of differentiation on Hox A5 expression was studied in COLO 205 cells treated with herbimycin A. The quantitative study showed that Hox A5 expression was increased when COLO 205 cells were induced to differentiate. The expression of Hox A5 was about 2-fold higher in the cells treated for 48 h compared to the untreated poorly-differentiated cells. The present study shows that Hox A5 may be involved in intestinal cell differentiation, in addition to its role in apoptosis.

Benzoquinones↗
PubMed1999

Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation.

Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Stem cell factor inhibits erythroid differentiation by modulating the activity of G1-cyclin-dependent kinase complexes: a role for p27 in erythroid differentiation coupled G1 arrest.

Terminal erythroid differentiation is accompanied by decreased expression of c-Kit and decreased proliferation of erythroid progenitor cells. Using a newly established erythroleukemia cell line HB60-5, which proliferates in response to erythropoietin (Epo) and stem cell factor (SCF) and differentiates when stimulated with Epo alone, we characterized several events associated with the cell cycle during erythroid differentiation. Forty-eight h after SCF withdrawal and Epo stimulation, there was strong inhibition of cyclin-dependent kinase (cdk) 4 and cdk6 activities, associated with an increase in the binding of p27 and p15 to cdk6. A significant increase in the binding of p27 to cyclin E- and cyclin A-associated cdk2 correlated with the inhibition of these kinases. In addition, the expression of c-Myc and its downstream transcriptional target Cdc25A were found to be down-regulated during Epo-induced terminal differentiation of HB60-5 cells. The loss of Cdc25A was associated with an increase in the phosphotyrosylation of cyclin E-associated cdk2, which may contribute to cell cycle arrest during differentiation. Although overexpression of p27 in HB60-5 cells caused G1 arrest, it did not promote terminal erythroid differentiation. Thus, the cell cycle arrest that involves p27 is part of a broader molecular program during HB60-5 erythroid differentiation. Moreover, we suggest that SCF stimulation of erythroblasts, in addition to inhibiting erythroid differentiation, activates parallel or sequential signals responsible for maintaining cyclin/cdk activity.

Animals↗
PubMed1992

Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers.

We have recently demonstrated that bufalin is a new potent inducer of the differentiation of human myeloid leukemia cells. The present work was carried out to examine further the effect of bufalin on the growth and characteristics of human leukemia-derived cell lines U937, ML1, and HL60. At concentrations of 5-10 nM, bufalin decreased the growth of ML1 cells preferentially at the G2 phase and U937 cells at the S and G2 phases of the cell cycle. Bufalin, under these conditions, induced the differentiation of U937, ML1, and HL60 cells to monocyte/macrophage-like cells by measuring the expression of various differentiation markers, as assessed by morphology and histochemistry, and ability to phagocytose latex particles, to reduce nitroblue tetrazolium, and to develop Fc receptors. U937 and ML1 cells started to differentiate at 4 and 6 h, respectively, after treatment with 10 nM bufalin and showed maximum differentiation 72 h later. At present, a mechanism for the bufalin-mediated induction of the differentiation of these human leukemia cells remains to be determined. The combination of bufalin with all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP16), or human gamma-interferon synergistically induced the differentiation of HL60 and U937 cells. A similar effect on ML1 cells was observed with the combination of bufalin with VP16 or human rTNF-alpha. These results suggest that bufalin in combination with VP16, all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, rTNF-alpha, or gamma-interferon may be very useful in the differentiation of human leukemia.

Antineoplastic Agents↗
PubMed1991

[Changes in various differentiation-related markers after differentiation induction of a pluripotent human embryonal carcinoma (EC) cell line by hexamethylene bisacetamide].

A pluripotent human EC cell line (NEC14) could be induced to morphologically differentiate by treatment with 10(-2) M HMBA for 3 days in vitro. The changes in various differentiation-related markers (cell surface antigens, lectin binding sites, intermediate filaments, secreted products and extracellular matrix proteins) after induction of differentiation were examined in order to clarify the differentiation lineage. The results were as follows: 1) The most conspicuous changes in cell surface antigens after differentiation were the expression of major human histocompatibility antigens (HLA-A,B,C) and the changes in stage specific embryonic antigens (SSEA-1-/SSEA-3(+)----SSEA-1+/SSEA-3-). 2) Vimentin, mesenchymal intermediate filament, was only detected after the differentiation. 3) Tenascin, an extracellular matrix protein produced in mesenchymal cells, was produced after the differentiation. These results indicate that HMBA can induce NEC14 cells to differentiate into mesenchymal elements of embryonal mesoderm.

Acetamides↗
PubMed1989

Two promoters that map to 5'-sequences of the human p53 gene are differentially regulated during terminal differentiation of human myeloid leukemic cells.

p53 is overexpressed in many transformed cells and expression of the gene is known to alter during terminal differentiation of cells in culture. Through analysis of recombinant vectors expressing the chloramphenicol acetyl transferase (CAT) gene we found that two promoters map to the 5'-portion of the human p53 oncogene. One promoter, p53p1, maps upstream of the non-coding first exon and the second, p53p2, maps within the first intron. By primer extension analysis of cellular RNA from a number of human cell lines, we found that p53p2 is a functional promoter in vivo. In order to test whether differential regulation of these promoters may be correlated with the control of expression of the p53 gene during differentiation, we have measured the activity of the two promoters by their ability to direct expression of the CAT gene during terminal differentiation of the human promyelocytic leukemia cell line HL-60. HL-60 cells stably harboring Epstein-Barr virus-derived recombinant plasmids that express the CAT gene from either p53p1 or p53p2 were induced to undergo terminal differentiation by a variety of chemical inducers to either granulocytes or monocytes and expression of the CAT gene was measured. The results indicate that while expression of p53p1 remained constant, expression from p53p2 was induced 5- to 10-fold during differentiation of these cells to either granulocytes or monocytes. Similarly, the endogenous p53p2 was found to be induced in HL-60 cells undergoing differentiation. Although the product of the p53p2 initiated transcript has not yet been characterized these results indicate that altered regulation of these two promoters may be important in modulating the expression of mRNA from this gene during terminal differentiation.

Cell Cycle↗
PubMed1985

Changes in glycosphingolipid composition during differentiation of human leukemic granulocytes in chronic myelogenous leukemia compared with in vitro granulocytic differentiation of human promyelocytic leukemia cell line HL-60.

Changes in glycosphingolipid (GSL) composition during differentiation of human leukemic granulocytes were investigated qualitatively and quantitatively in immature and mature granulocytic cells derived from human chronic myelogenous leukemia (CML) cases and were compared with those found in the in vitro granulocytic differentiation of the human promyelocytic leukemia HL-60 cell line. Two neutral GSLs, ceramide monohexoside and ceramide dihexoside, and two molecular species of gangliosides, one being the ganglio-series ganglioside NeuAc(alpha 2-3)Gal(beta 1-4)Glc-Cer (GM3) and the other being the lacto-series sialosylparagloboside, were predominant in the granulocytic cells at an early maturation stage. During the granulocytic differentiation of CML cells, the contents of ceramide dihexoside and paragloboside increased strikingly with a concomitant decrease in ceramide monohexoside, and the total amount of neutral GSLs increased to about three times as much as that of the most immature granulocytic cells, myeloblasts. On the other hand, lacto-series gangliosides, with longer sugar moieties increased with a concomitant decrease in ganglio-series ganglioside GM3, and the ganglioside profile became more complex. The total content of ganglioside increased in parallel with the complexity of the ganglioside profile. Similar differentiation-associated changes were also found in GSL composition during the in vitro granulocytic differentiation of HL-60 cells. However, a marked difference between the differentiation-dependent change in the GSL composition of CML cells and that of HL-60 cells was observed for a ganglioside species which was found to be one of the major gangliosides in normal neutrophils: in the former, the ganglioside level increased up to the level in normal mature granulocytes as the cells differentiated; in contrast, it decreased significantly during granulocytic differentiation of the latter cells. When the GSL composition of the neutrophils obtained from CML cells, which were apparently normal as to morphology, stimulus-induced membrane potential changes, and superoxide-producing capacity, was compared with that of normal neutrophils, an obvious difference was observed between them, especially with regard to ganglioside GM3; the amount of ganglioside GM3 in the former was about one-sixth of that in the latter. This finding indicates some alterations in the cell membrane structure of neutrophils of CML origin.

Carbohydrate Metabolism↗
PubMed1995

Induction of differentiation of WEHI-3B D+ leukemic cells transfected with differentiation-stimulating factor/leukemia inhibitory factor receptor cDNA.

Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor can induce the differentiation of mouse myeloid leukemia M1 cells and also stimulate proliferation of the interleukin-3 (IL-3)-dependent cell line, DA-1a. To determine whether D-factor can induce the differentiation of leukemia cells other than M1 cells, WEHI-3B D+ mouse myelomonocytic leukemia cells were transfected with a plasmid containing mouse D-factor receptor cDNA. Expression of D-factor receptor in transfected cells was determined by binding of [125]D-factor and analyzed by Scatchard's method. The transfected cells had high-affinity D-factor receptors with a dissociation constant of 100 to 200 pmol/L and binding sites per cell varied from 67 to 1,500 among several clones. The cells expressing a high level of D-factor receptor were induced to differentiate by D-factor; about 60% of the cells exhibited the ability to reduce nitroblue tetrazolium and expression of the differentiation antigen Mac-1 (CD11b) on the cell surface increased. The effect of cytokines, which induce the differentiation of M1 cells, on the transfected WEHI-3B cells was examined. The sensitivity to oncostatin M was identical to that against D-factor in the cells of each clone. Expression of D-factor receptor in WEHI-3B cells promoted sensitivity to IL-6 and granulocyte colony-stimulating factor (G-CSF). Induction of differentiation of the cells accompanied the suppression of proliferation. Treatment of the cells with D-factor for longer than 5 days resulted in 50% inhibition of growth. These results indicate that the stimulating effect of D-factor on the differentiation of malignant myeloid cells is not unique to M1 cells.

Animals↗
PubMed1994

Characterization of conventional protein kinase C (PKC) isotype expression during F9 teratocarcinoma differentiation. Overexpression of PKC alpha alters the expression of some differentiation-dependent genes.

F9 teratocarcinoma is a useful model for studying early embryogenesis since these cells can differentiate into primitive or parietal endoderm under the influence of retinoic acid or retinoic acid and cyclic AMP, respectively. We have found that three isoforms of protein kinase C (PKC alpha, -beta, and -gamma) were expressed in undifferentiated stem cells. When the cells were treated with retinoic acid either alone or in the presence of cAMP for 120 h, PKC alpha mRNA and protein levels increased, whereas those of PKC beta and PKC gamma became undetectable. These changes began within 24 h of drug treatment and were complete by 48-72 h. In order to determine the functional significance of the induction of PKC alpha during F9 differentiation, we established two stable transfectants that overexpressed PKC alpha protein between 4- and 5-fold compared to wild type cells. Characterization of these cell lines revealed an altered pattern of expression of some of the markers of F9 differentiation. The clone that had the highest amount of PKC alpha protein constitutively expressed mRNA for type IV collagen and c-Jun, which are not normally expressed until 24-48 h of treatment with differentiation agents. In the other overexpressing clone, these markers were induced much faster than in wild type cells. The growth rate of both overexpressing clones was less than wild type cells, while the expression of the PKC beta protein in these clones was similar to the levels found in differentiated F9 cells. However, other markers of differentiation, including the cellular morphology and levels of pST6-135 and c-myc RNA, responded to agents identically in both wild type and PKC-alpha-overexpressing clones. Therefore, overexpression of PKC alpha is not sufficient to induce full differentiation of F9 cells. However, our data suggest that certain pathways that lead to the expression of differentiation-dependent genes are regulated by PKC alpha protein levels.

Animals↗
PubMed1996

Evidence for distinction of the differentiation-inducing activities and cytostatic properties of 9-(2-phosphonylmethoxyethyl)adenine and a variety of differentiation-inducing agents in human erythroleukemia K562 cells.

The acyclic nucleoside phosphonate derivative 9-(2-phosphonylmethoxyethyl) adenine (PMEA) is a strong inducer of differentiation in several tumor cell lines, including human erythroleukemia K562 cells. A PMEA-resistant K562 cell line (designated K562/PMEA-1) was selected in the presence of escalating PMEA concentrations. This cell line proved to be insensitive to the induction of erythroid differentiation by PMEA. It also was 108-fold resistant to the cytostatic effects of PMEA. The K562/PMEA-1 cells showed reduced sensitivity to the differentiation-inducing capacity and cytostatic activity of several closely related PMEA analogs. Furthermore, the mutant cells exhibited a decreased sensitivity to the differentiation-inducing activity of a wide variety of structurally nonrelated antimetabolites targeted at different enzymes of nucleotide biosynthesis. A 5-25-fold higher concentration of each of these compounds was required to obtain the same level of differentiation in the K562/PMEA-1 cells as in the wild-type cells. However, unlike the PMEA derivatives, the antimetabolites remained equally cytostatic for the mutant K562 cells and for the wild-type cells. Our results reveal two unique features of the K562/PMEA-1 cells: (i) specific resistance to both the differentiation-inducing and cytostatic effects of several acyclic nucleoside phosphonate analogs (which can be accounted for by a diminished cellular uptake and subsequent metabolism of the compounds) and (ii) nonspecific resistance to antimetabolites with regard to their differentiation-inducing, but not cytostatic, properties (which must reside in an unspecified alteration of a common site of the differentiation process that is shared by all of these antimetabolites).

Adenine↗
PubMed1997

A decrease in the intracellular guanosine 5'-triphosphate concentration is necessary for granulocytic differentiation of HL-60 cells, but growth cessation and differentiation are not associated with a change in the activation state of Ras, the transforming principle of HL-60 cells.

We found that when human promyelocytic leukemic cells (HL-60 cells) were induced to differentiate along the granulocytic lineage by two diverse mechanisms (starvation for an essential amino acid or treatment with DMSO), there was a marked decrease in the intracellular guanosine 5'-triphosphate (GTP) concentration with no change in the guanosine 5'-diphosphate (GDP) concentration. Differentiation was prevented by guanine or guanosine in a dose-dependent manner. We showed that: (a) guanine had to be converted to a nucleotide because it did not prevent differentiation of hypoxanthine-guanine phosphoribosyltransferase-deficient HL-60 cells; (b) the effect of guanine correlated with a return of the cytosolic GTP:GDP ratio to normal; and (c) other purine bases were not effective. We hypothesized that the decreased GTP:GDP ratio in differentiating HL-60 cells might decrease the relative amount of GTP bound to Ras, a key regulatory GTP-binding protein important to cell growth and differentiation. Consistent with data showing that HL-60 cells harbor an activating N-Ras mutation, we found that the percentage of Ras molecules in the GTP-bound state was high in proliferating HL-60 cells (27 +/- 3%) compared with other cultured mammalian cells (< 1%); however, we found no change in the activation state of Ras when cells ceased to proliferate and differentiated in response to DMSO, amino acid deprivation, or inhibitors of guanylate synthesis. We conclude that: (a) a decrease in the intracellular GTP concentration is necessary for HL-60 cells to undergo granulocytic differentiation; and (b) although a high degree of Ras activation contributes to the malignant phenotype of the cell, there is no change in the activation state of Ras during granulocytic differentiation.

Amino Acids↗
PubMed1999

Identification of true differentially expressed mRNAs in a pair of human bladder transitional cell carcinomas using an improved differential display procedure.

Differential display in combination with arbitrarily primed polymerase chain reaction (PCR) fingerprinting has become one of the most powerful techniques to identify and isolate mRNAs that are differentially expressed in pairs of biological samples. However, in many cases the cDNA band corresponding to the differentially amplified product contains several cDNA species that comigrate with the cDNA of interest due to the poor resolution of the fingerprinting gels, thus hampering further analysis and identification of the desirable cDNA. To improve the electrophoretic resolution of differentially amplified cDNAs, we have utilized Resolver Gold agarose gel electrophoresis (Ingenius) as an additional step to overcome downstream problems encountered during RNA fingerprinting experiments. To illustrate the power of the modified differential display procedure we present a detailed analysis of the cDNA products differentially displayed in tumor biopsies obtained from a noninvasive (grade II, Ta) and an invasive (grade III, T2-T4) human bladder transitional cell carcinoma (TCC). Several genes that were differentially expressed in this tumor pair were identified. These included: tropomyosin 4, the protein disulfide isomerase precursor (PDI), MRP14, signal transducer CD24, keratins 8 and 13, cytochrome oxidase subunit IV (COXIV), putative transcription factor HOX-1.3, as well as two novel genes of yet unknown function. All of the identified cDNAs were shown to be truly differentially expressed by Northern blotting, reverse transcriptase-PCR (RT-PCR), and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of the corresponding lesions.

Bisbenzimidazole↗
PubMed1998

The nonradioisotopic representation of differentially expressed mRNA by a combination of RNA fingerprinting and differential display.

In many applications, an understanding of differentially expressed genes in different tissues or owing to an applied stimulus is important. However, the wide use of two rather similar polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR [RAP-PCR] and differential display [DDRT-PCR]) has shown that reproducibility is still a problem. By combining features of both RAP-PCR and DDRT-PCR, a technique has recently been developed that avoids some of the disadvantages, but the use of radioisotopes for band detection still limits its application. We have improved this technique for analyzing differentially expressed mRNA by resolving the amplified products on nondenaturing polyacrylamide gels and subsequently staining the gels with silver nitrate. Our modification allows the identification of differentially expressed bands with a very high accuracy. Therefore these bands can be very easily reamplified and sequenced directly. Subsequently the differential expression can be verified by semiquantitative RT-PCR with specific primers derived from sequence data. These improvements, together with nonradioactive sequencing techniques, make it possible to do DD analysis completely without a health hazardous owing to radioactivity. The nonradioisotopic differentially expressed mRNA-PCR (DEmRNA-PCR) is a reliable and useful modification of available differential expression methods.

Cells, Cultured↗
PubMed1996

Differential modulation by interferon gamma of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens.

Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens-present also in normal melanocytes-and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon gamma (IFN gamma) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFN gamma, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-gamma induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-gamma plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells.

Animals↗
PubMed2003

Dietary factors and the risk of gastric cancer among Japanese women: a comparison between the differentiated and non-differentiated subtypes.

PURPOSE: Using data from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC), a case-referent study was performed to clarify whether dietary habits differentially impact on the risk of female gastric cancers of different histological subtypes. METHODS: The study subjects comprised 508 histologically confirmed female gastric cancer cases (156 differentiated, 352 non-differentiated), identified via hospital cancer registry and surgical records between 1988 to 1998. The referents were 36,490 cancer-free first-visit female outpatients over 30 years old presenting at the center within the same period of time. The odds ratios were estimated by logistic regression analyses and adjusted for potential confounding factors. RESULTS: Frequent vegetable and fruit conferred reduced risk of both differentiated and non-differentiated female gastric cancers in similar patterns. In addition, reduced risk was observed for intake of fish and soybean products, particularly for the non-differentiated type. CONCLUSIONS: These results suggest that both differentiated and non-differentiated female gastric cancers are preventable by frequent intake of vegetable and fruit, fish and soybean products, suggested to be common protective factors, although the possibility of some variation in the impact of the diet on different histopathological entities needs to be further addressed.

Adult↗
PubMed2004

Comparative immunohistochemical analysis of granulosa and sertoli components in ovarian sex cord-stromal tumors with mixed differentiation: potential implications for derivation of sertoli differentiation in ovarian tumors.

Granulosa cell tumors of the ovary occasionally show admixed Sertoli components, just as tumors that are predominantly Sertoli or Sertoli-Leydig cell tumors can contain minor granulosa elements. Although the immunoprofiles of pure granulosa cell tumors and pure Sertoli cell tumors have been characterized, little is known regarding what immunophenotypic relationships exist between the granulosa and Sertoli components in ovarian sex cord-stromal tumors that contain both elements. Furthermore, it is not completely understood why sex cord-stromal tumors of the ovary with female-type (granulosa) differentiation can produce male-type (Sertoli) differentiation. To better understand why simultaneous differentiation into female-type and male-type components occurs, eight tumors with mixed differentiation were stained with a panel of antibodies to androgen receptor (AR), calretinin, CD10, CD99, estrogen receptor, inhibin, Ki-67, low molecular weight cytokeratin, pancytokeratin, progesterone receptor, p53, and vimentin. Immunohistochemical composite scores were determined separately for the matched pairs of granulosa and Sertoli components in each case. Differences between both components were statistically analyzed using the Wilcoxon signed rank test. AR and vimentin expression showed a difference at the 10% statistical significance level (p < 0.1), demonstrating higher levels of expression in the granulosa components. The differences between the granulosa and Sertoli components in expression of CD99, inhibin, or pancytokeratin were not statistically significant (p > 0.1, each). Statistical calculations could not be made for calretinin, CD10, estrogen receptor, Ki-67, low molecular weight cytokeratin, progesterone receptor, or p53, although the overall mean levels of expression of CD10 and low molecular weight cytokeratin were substantially higher in the Sertoli components. Not surprisingly, the granulosa and Sertoli components of ovarian sex cord-stromal tumors with mixed differentiation show overlapping immunophenotypic profiles consistent with derivation from a common lineage rather than reflecting a composite tumor. However, because components of a sex cord-stromal tumor simultaneously differentiate along granulosa or Sertoli lines, they seem to show preferential expression of certain antigens. CD10 and low molecular weight cytokeratin are more often associated with Sertoli cell differentiation, whereas AR and vimentin expression seem to reflect granulosa differentiation.

Cell Transformation, Neoplastic↗
PubMed2007

Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors.

Mesenchymal stem cells (MSCs) are a candidate for replacing chondrocytes in cell-based repair of cartilage lesions. However, it has not been clarified if these cells can acquire the hyaline phenotype, and whether chondrocytes and MSCs show the same expression patterns of critical control genes in development. In order to study this, articular chondrocytes and iliac crest derived MSCs were allowed to differentiate in pellet mass cultures. Gene expression of markers for the cartilage phenotype, helix-loop-helix (HLH) transcription factors, and chondrogenic transcription factors were analyzed by real-time PCR. Matrix production was assayed using biochemical analysis for hydroxyproline, glycosaminoglycans, and immunohistochemistry for collagen types I and II. Significantly decreased expression of collagen type I was accompanied by increased expression of collagen types IIA and IIB during differentiation of chondrocytes, indicating differentiation towards a hyaline phenotype. Chondrogenesis in MSCs on the other hand resulted in up-regulation of collagen types I, IIA, IIB, and X, demonstrating differentiation towards cartilage of a mixed phenotype. Expression of HES1 increased significantly during chondrogenesis in chondrocytes while expression in MSCs was maintained at a low level. The HLH gene HES5 on the other hand was only detected in chondrocytes. Expression of ID1 decreased significantly in chondrocytes while the opposite was seen in MSCs. These findings suggest that chondrocytes and MSCs differentiated and formed different subtypes of cartilage, the hyaline and a mixed cartilage phenotype, respectively. Differentially regulated HLH genes indicated the possibility for HLH proteins in regulating chondrogenic differentiation. This information is important to understand the potential use of MSCs in cartilage repair.

Adult↗
PubMed2006

Growth and differentiation factor-5 (GDF-5) stimulates osteogenic differentiation and increases vascular endothelial growth factor (VEGF) levels in fat-derived stromal cells in vitro.

Fat-derived adult mesenchymal stem cells can differentiate into different phenotypes reflecting their potential to regenerate various skeletal tissues. These properties together with the association of adipose with skeletal tissues formed the basis of our study to establish an experimental model for using fat-derived stromal cells to undergo osteogenic differentiation in vitro under the influence of either growth and differentiation factor-5 (GDF-5) or bone morphogenetic protein-2 (BMP-2). Members of the BMP/GDF family of proteins are known for their ability to elicit skeletal morphogenesis, but little is known about the mechanism whereby these morphogens exert their effect on the osteogenic differentiation of fat-derived stromal cells. We compared the effects of GDF-5 and BMP-2 in their recombinant forms to qualitatively and quantitatively determine their influence on the osteogenic differentiation of fat derived stromal cells by examining the effects on mineralization, extracellular matrix, cell proliferation, biochemistry, and gene expression. We identified that GDF-5 not only promotes osteogenic differentiation of rat fat-derived stromal cells, but also may promote angiogenic activity of stromal cells by increasing vascular endothelial growth factor (VEGF) gene expression in vitro. These data suggest that several distinct regulatory mechanisms may exist in association with osteogenic differentiation.

Adipocytes↗