Ibandronate increases the expression of the pro-apoptotic gene FAS by epigenetic mechanisms in tumor cells.

There is growing evidence that aminobisphosphonates like ibandronate show anticancer activity by an unknown mechanism. Biochemically, they prevent posttranslational isoprenylation of small GTPases, thus inhibiting their activity. In tumor cells, activated RAS-GTPase, the founding member of the gene family, down-regulates the expression of the pro-apoptotic gene FAS via epigenetic DNA-methylation by DNMT1. We compared ibandronate treatment in neoplastic human U-2 osteosarcoma and in mouse CCL-51 breast cancer cells as well as in the immortalized non-neoplastic MC3T3-E1 osteoblastic cells. Ibandronate attenuated cell proliferation in all cell lines tested. In the neoplastic cells we found up-regulation of caspases suggesting apoptosis. Further we found stimulation of FAS-expression as a result of epigenetic DNA demethylation that was due to down-regulation of DNMT1, which was rescued by re-isoprenylation by both geranylgeranyl-pyrophosphate and farnesylpyrophosphate. In contrast, ibandronate did not affect FAS and DNMT1 expression in MC3T3-E1 non-neoplastic cells. Data suggest that bisphosphonates via modulation of the activity of small-GTPases induce apoptosis in neoplastic cells by DNA-CpG-demethylation and stimulation of FAS-expression. In conclusion the shown epigenetic mechanism underlying the anti-neoplastic activity of farnesyl-transferase-inhibition, also explains the clinical success of other drugs, which target this pathway.

Epigenetic mechanisms in anti-cancer actions of bioactive food components--the implications in cancer prevention.

The hallmarks of carcinogenesis are aberrations in gene expression and protein function caused by both genetic and epigenetic modifications. Epigenetics refers to the changes in gene expression programming that alter the phenotype in the absence of a change in DNA sequence. Epigenetic modifications, which include amongst others DNA methylation, covalent modifications of histone tails and regulation by non-coding RNAs, play a significant role in normal development and genome stability. The changes are dynamic and serve as an adaptation mechanism to a wide variety of environmental and social factors including diet. A number of studies have provided evidence that some natural bioactive compounds found in food and herbs can modulate gene expression by targeting different elements of the epigenetic machinery. Nutrients that are components of one-carbon metabolism, such as folate, riboflavin, pyridoxine, cobalamin, choline, betaine and methionine, affect DNA methylation by regulating the levels of S-adenosyl-L-methionine, a methyl group donor, and S-adenosyl-L-homocysteine, which is an inhibitor of enzymes catalyzing the DNA methylation reaction. Other natural compounds target histone modifications and levels of non-coding RNAs such as vitamin D, which recruits histone acetylases, or resveratrol, which activates the deacetylase sirtuin and regulates oncogenic and tumour suppressor micro-RNAs. As epigenetic abnormalities have been shown to be both causative and contributing factors in different health conditions including cancer, natural compounds that are direct or indirect regulators of the epigenome constitute an excellent approach in cancer prevention and potentially in anti-cancer therapy.

Relationship between carnitine, fatty acids and insulin resistance.

Increased plasma free fatty acid (FFA) levels are a feature of insulin resistance and type 2 diabetes. The aim of the present study was to assess the effect of L-carnitine supplementation on plasma lipids and the expression of enzymes in peripheral mononucleated cells (PMNC) involved in the regulation of fatty acid and glucose oxidation. L-Carnitine supplementation of 2 g/day resulted in a significant decrease in plasma FFA and in a less pronounced diminution of the plasma triacylglycerols. In addition, a concomitant increase in the relative mRNA abundances of carnitine acyltransferases (5- to 10-fold) and of the carnitine carrier OCTN2 (12-fold) in PMNC of pregnant women was found. The results of the present study provide evidence that L-carnitine supplementation in pregnancy (2 g/day) avoids a striking increase in plasma FFA, which are thought to be the main cause of insulin resistance and consequently gestational diabetes mellitus.

Lysyl oxidase (LOX) mRNA expression and genes of the differentiated osteoblastic phenotype are upregulated in human osteosarcoma cells by suramin.

It is well known that suramin influences proliferation and differentiation of tumour cells. To study whether and how suramin effects osteosarcoma (OS) cells, proliferation, differentiation, LOX mRNA expression and telomerase activity (TA) was analysed in the human MG-63 and U-2 OS, and the rat UMR-106 OS cell lines. Data show that suramin inhibited proliferation in the human cell lines and upregulated alkaline phosphatase activity. TA was attenuated in the human cells while in UMR-106 it was not changed. In UMR-106 suramin had no influence on osteocalcin and LOX expression, in the human cells however, both genes were upregulated.

Recursive causality in evolution: a model for epigenetic mechanisms in cancer development.

Interactions between adaptative and selective processes are illustrated in the model of recursive causality as defined in Rupert Riedl's systems theory of evolution. One of the main features of this theory also termed as theory of evolving complexity is the centrality of the notion of 'recursive' or 'feedback' causality - 'the idea that every biological effect in living systems, in some way, feeds back to its own cause'. Our hypothesis is that "recursive" or "feedback" causality provides a model for explaining the consequences of interacting genetic and epigenetic mechanisms which are known to play a key role in development of cancer. Epigenetics includes any process that alters gene activity without changes of the DNA sequence. The most important epigenetic mechanisms are DNA-methylation and chromatin remodeling. Hypomethylation of so-called oncogenes and hypermethylation of tumor suppressor genes appear to be critical determinants of cancer. Folic acid, vitamin B12 and other nutrients influence the function of enzymes that participate in various methylation processes by affecting the supply of methyl groups into a variety of molecules which may be directly or indirectly associated with cancerogenesis. We present an example from our own studies by showing that vitamin D3 has the potential to de-methylate the osteocalcin-promoter in MG63 osteosarcoma cells. Consequently, a stimulation of osteocalcin synthesis can be observed. The above mentioned enzymes also play a role in development and differentiation of cells and organisms and thus illustrate the close association between evolutionary and developmental mechanisms. This enabled new ways to understand the interaction between the genome and environment and may improve biomedical concepts including environmental health aspects where epigenetic and genetic modifications are closely associated. Recent observations showed that methylated nucleotides in the gene promoter may serve as a target for solar UV-induced mutations of the p53 tumor suppressor gene. This illustrates the close interaction of genetic and epigenetic mechanisms in cancerogenesis resulting from changes in transcriptional regulation and its contribution to a phenotype at the micro- or macroevolutionary level. Above-mentioned interactions of genetic and epigenetic mechanisms in oncogenesis defy explanation by plain linear causality, things like the continuing adaptability of complex systems. They can be explained by the concept of recursive causality and has introduced molecular biology into the realm of cognition science and systems theory: based on the notion of so-called feedback- or recursive causality a model for epigenetic mechanisms with relevance for oncology and biomedicine is provided.

Do blood cells mimic gene expression profile alterations known to occur in muscular adaptation to endurance training?

Exercise is known to upregulate mRNA synthesis for carnitine palmitoyl transferase1 (CPT1) and possibly also other mitochondrial carnitine acyltransferases in muscle tissue. The aim of this study was to test whether such an adaptation of oxidative metabolism in skeletal muscle is a systemic process and consequently, also affects other cells. Messenger RNA levels of five genes [carnitine palmitoyl transferases 1 and 2 (CPT1 and CPT2), carnitine acetyltransferase (CRAT), carnitine palmitoyltransferase 2 (CPT2), microsomal carnitine palmitoyltransferase (GRP58) and organic cation transporter (OCTN2)] were determined with quantitative real time polymerase chain reaction (PCR) in blood cells and in muscle biopsy samples from six cross country skiers before and 6 months after a high volume/low intensity exercise training, when training had elicited a significantly slower rate of lactate accumulation. Quantitative real time PCR showed that levels of mRNA in blood cells correlated significantly (CPT1B: P< 0.001) with those in muscle tissue from the same donors. After 6-months training, there was a 15-fold upregulation of CPT1B mRNA, a six to ninefold increase of CRAT mRNA, of CPT2 mRNA, GRP58 mRNA, and of OCTN2 mRNA. The observation of a concordant stimulation of CPT1, CPT2, CRAT, GRP58 and OCTN2 transcription in blood cells and muscle tissue after 6-month-endurance training leads the hypothesis of a common stimulation mechanism other than direct mechanical stress or local chemical environment, but rather humoral factors.

Expression of insulin-like growth factor-I (IGF-I) in aneurysmal bone cyst.

The effects of insulin-like growth factor-I on bone tissue and its role in bone development have been extensively investigated, but there is little information on its role in the pathogenesis of aneurysmal bone cyst. Therefore, using the techniques of immunohistochemistry and in situ hybridization, the authors studied the expression of insulin-like growth factor-I in 19 specimens of aneurysmal bone cyst. Insulin-like growth factor-I or specific mRNA sequences encoding for insulin-like growth factor-I were detectable in all specimens tested and were mainly localized in multinucleate giant cells. In contrast, only insignificant levels of insulin-like growth factor-I expression were detectable in normal human bone tissue. Taken together with the previously reported role of insulin-like growth factor-I in the pathogenesis of giant cell tumor, the findings of this study suggest that insulin-like growth factor-I may play a role in the pathogenesis of aneurysmal bone cyst.

Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML).

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.

Quantitative analysis of immune-mediated stimulation of tumor necrosis factor-alpha in macrophages measured at the level of mRNA and protein synthesis.

Expression of cytokines such as tumor necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS) has been associated with inflammatory and regulatory immune reactions. Antigen-presenting cells, especially macrophages, play a central role in directing immune responses by synthesizing different cytokines. For the analysis of cytokine synthesis, we compared quantitative changes in mRNA and protein synthesis of TNF-alpha in RAW 264.7 cells stimulated with 0.1 ng/ml LPS. TNF-alpha mRNA was quantified using the LightCycler SYBR Green technology (Idaho Technology, Inc., Salt Lake City, Utah, USA). RAW 264.7 cells showed a basal TNF-alpha mRNA expression which increased approximately sixfold after 2 h of stimulation with LPS. TNF-alpha synthesis was analyzed at the protein level using a mouse-specific sandwich enzyme-linked immunosorbent assay (ELISA) and indicated a 56-fold increase in TNF-alpha protein concentration after 4 h. Thus, real-time polymerase chain reaction (PCR) is a sensitive and rapid method for quantitative determination of LPS-induced TNF-alpha expression. However, it requires extremely robust reaction parameters to be reliable for accurate quantification.

Hematopoietic recovery after IEV chemotherapy for malignant lymphoma followed by different cytokines can be monitored by analysis of Galpha 16 and CD34.

The hematopoiesis-specific G protein alpha subunit Galpha16 is a specific element in the signal transduction of the early hematopoietic cytokine network. As Galpha16 mRNA can be detected in early hematopoietic progenitor cells, RT-PCR for Galpha16 can be used as a sensitive marker of hematopoietic activity. The aim of this study was to test the possible use of Galpha16 determinations for monitoring cytokine effects on hematopoietic recovery after chemotherapy in patients. We correlated presence of Galpha16 mRNA and CD34 surface antigen with hematopoietic recovery in six lymphoma patients undergoing salvage therapy with different cytokine support (IEV followed by G-CSF, IL-3, or placebo). Regardless of different cytokine schedules with different time courses, hematopoietic recovery was always preceded by transcription of Galpha16. Monitoring the expression of Galpha16 mRNA by RT-PCR is a highly sensitive diagnostic tool for analyzing hematopoietic recovery after chemotherapy and for characterizing the effects of cytokines on hematopoiesis.

The hemoregulatory peptide pEEDCK may inhibit stem cell proliferation via hydropathic binding to antisense sequence motifs in interleukin-11 and other growth factors.

In undisturbed bone marrow, most hemopoietic stem cells are nonproliferating despite the presence of multiple growth factors. Endogenous inhibitory factors are responsible for maintenance of this quiescence. Previously we sequenced and synthesized the inhibitory pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK), which originally derives from granulocytes, and investigated the role of this peptide in stem cell quiescence. To provide some mechanistic insight, in the present work we studied the structural relationship of this peptide to specific growth-factor-derived sequence motifs. In the murine system in vivo as well as in long-term bone marrow, antiserum to pEEDCK produced a significant stimulation of formation of colony-forming units-granulocyte/macrophage. Binding of peptides to proteins often takes place at hydropathically complementary sites. Therefore, we searched for peptides corresponding to the complementary sequence to pEEDCK. We identified antisense sequences in the genes of various cytokines and cytokine receptors including interleukin-11. The corresponding peptide Val-Leu-Leu-Thre-Arg (VLLTR) and several other peptides hydropathically complementary to pEEDCK were synthesized. We found that pEEDCK binds specifically to these peptides as well as to complete interleukin-11. Dissociation constants were in the 10 microM range. The peptide hydropathically corresponding to pEEDCK (VLLTR) was found to stimulate colony-forming units-granulocyte/macrophage formation. Our data suggest that pEEDCK could exert a coordinating function in the hemopoietic cytokine network by binding to multiple regulatory proteins and modulating their activity.

In vivo and in vitro effects of cytokines and the hemoregulatory peptide dimer (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 on G alpha16-positive hematopoiesis.

G proteins play an important role in signal transduction from cytokine receptors to intracellular effectors via different pathways, eg involving tyrosine kinases. In our previous studies, we demonstrated that mRNA expression of the hematopoiesis-specific G protein alpha-subunit G alpha16 is a sensitive marker indicating the appearance of early myeloid and lymphoid progenitors. This study was designed to investigate cytokine effects on hematopoiesis in vivo and in vitro as reflected by G alpha16 expression and sensitivity to the hemoregulatory peptide (pEEDCK)2 which harbors a structural homology to the effector domain of G alpha16. Investigations on blood samples from lymphoma patients undergoing salvage therapy with different cytokine support showed that monitoring of the expression of G alpha16 mRNA which appears to play a role in cytokine signalling via tyrosine kinases was a valuable complementation to CD34 screening for analyzing hematopoietic recovery after chemotherapy. We demonstrated that in contrast to CD34 which is only expressed in quiescent cells, G alpha16 transcription occurs independently of cell cycle state. In vitro, we could show that G alpha16 was also a valuable marker for confirming the immature state of ex vivo expanded blood stem cells from patients. A further part of the study was focused on the response of G alpha16 and CD34 expressing cells to the granulocyte-derived hemoregulatory peptide (pyroGlu-Glu-Asp-Cys-Lys)2 = (pEEDCK)2 which harbors a G alpha16-homologous sequence motif. Results obtained from in vitro assays which involved estimation of colony outgrowth from CD34-positive cells showed that the effect of (pEEDCK)2 on CD34 cells enhanced the effect of IL-3 or SCF. These data indicate that G alpha16 may co-operate with (pEEDCK)2 in triggering the cytokine response of immature hematopoietic cells.

Monitoring of hematopoietic recovery after autologous stem cell transplantation by analysis of G alpha 16 mRNA and CD34 surface glycoprotein.

The hematopoiesis-specific G protein alpha subunit G alpha16 was shown to be expressed in early normal and malignant hematopoietic cell lines and has been suggested to play an important role in signal transduction of hematopoiesis. We previously demonstrated a strict correlation of G alpha16 mRNA and CD34 antigen expression in peripheral blood stem cells (PBSC). In PBSC mobilization, both markers are detectable at the time of hematopoietic recovery and progenitor cell release. In this study the possible use of G alpha16 determination in peripheral blood samples for monitoring patients undergoing stem cell transplantation was investigated. Normal peripheral blood is negative for G alpha16 expression. In all five patients G alpha16 mRNA expression appeared shortly before the time of blood cell recovery. When tested together with CD34 (three cases) a pattern different from CD34 antigen expression was found, reflecting a different mechanism of action. In two cases with different time points of leukocyte and platelet recovery G alpha16 mRNA was detected at both time points but not in the interval, thus suggesting a role of G alpha16 in multipotent precursor cells. CD34 mRNA tested in three patients was not detected at any time; this argues for different regulation of CD34 and G alpha16 mRNA. G alpha16 may be used as an indicator of hematopoietic recovery after autologous stem cell transplantation, suggesting that there are cell type-specific G protein-mediated signal transduction pathways of early hematopoiesis.

Association of human T-cell leukemia virus and myelodysplastic syndrome in a central European population.

In addition to a few disorders such as acute T-cell leukemia that are typically associated with the human T-cell leukemia virus (HTLV) 1 in endemic regions, this virus may also play a role in some other hematological diseases. Here, we examine the incidence of HTLV in hematological diseases from a nonendemic region in central Europe. Data obtained by PCR and/or serological techniques from a total of 730 cases showed that besides the expected presence of HTLV-1 in T-lymphoid diseases (2 of 27 cases), HTLV-1 was only detected in myelodysplastic syndrome (MDS), in which an incidence of 17% (11 of 65 cases) was found. A correlation with a history of multiple transfusions or treatment with blood products in the HTLV-1-positive MDS could not be ascertained. Cytogenetics detected the presence of del(5)(q) in six HTLV-positive cases (five MDS and one T-cell acute lymphocytic leukemia) but in only one HTLV-negative case. These data indicate that allelic deletions of a series of 5q-located genes that typically occur in MDS may be associated with HTLV infections in central Europe.

Expression of G alpha 16, a G-protein alpha subunit specific for hematopoiesis in acute leukemia.

G-proteins are essential in signal transduction pathways. A G-protein alpha subunit termed G alpha 16 was found to be exclusively expressed in hematopoietic cell lines. In cells derived from patients, G alpha 16 expression has been detected in progenitor- and pre-B ALL cells and also in peripheral blood stem cells (PBSC). In this study, we analyzed G alpha 16 expression using a RT-PCR technique by testing elutriated blood cells from normal donors, PBSC from breast cancer patients and bone marrow or peripheral blood cells from acute leukemia patients. Both of two ALL patients and 15/16 AML patients expressed G alpha 16. In elutriation experiments, G alpha 16 expression was found in fractions containing the highest number of precursor cells but was absent in mature T and B cell fractions. In addition, CD34-enriched PBSC were positive for G alpha 16 expression. Further in vitro experiments using the cell line KG1 showed that G alpha 16 expression was not affected by the growth inhibiting hemoregulatory peptide pEEDCK which has a sequence homology present within G alpha 16. Taken together, these data demonstrate that G alpha 16 is expressed in various normal and malignant hematopietic progenitors but not in their differentiated counterparts. G alpha 16 could play a vital role in signal transduction pathways controlling proliferation in early normal and malignant hematopoiesis.

Cytogenetic findings in 175 patients indicate that items of the Kiel classification should not be disregarded in the REAL classification of lymphoid neoplasms.

Cytogenetics have proved to be a valuable tool for classifying systemic lymphatic neoplasms, as this technique allows different stem line aberrations and clonal developments to be distinguished. This study was designed to analyze how far groups defined according to common cytogenetic features correlated with their position in either the Kiel (KC) or the REAL classification. Cytogenetic analyses were performed on material from 175 patients with lymphoid neoplasms (LN). Samples were prepared from peripheral blood and bone marrow in acute lymphoblastic leukemia (ALL), from bone marrow in multiple myeloma (MM), and from lymph node biopsies in lymphomas. The results of this study support the inclusion of ALL, MM, and extranodal lymphomas into a comprehensive classification, because their chromosomal aberrations were always characteristic for LN. From the cytogenetic point of view, a subgroup of ALL appears as a leukemic manifestation of lymphoblastic lymphoma. MM have structural aberrations of chromosomes 1, 11, and 14 and secondary aberrations of chromosomes 3, 6, 7, 12, 13, and 18, all of which are characteristic for lymphatic disease. The groups with follicle center cell lymphoma and mantle cell lymphoma correlate well with our results both in the low-grade subtype and in the blastic variant type, the majority of cases demonstrating t(14; 18) and its variants and t(11; 14), respectively. In contrast, the group of diffuse large B-cell (DLB) lymphomas proved to be heterogeneous on the basis of our cytogenetic results. Accordingly, we would suggest keeping the immunoblastic lymphoma (IB) subtype defined by the KC. IB demonstrates no stem line aberration in common with any other group and seems to be characterized by stem line aberrations involving chromosomes 3 and 6. As some DLB lymphomas have a t(14;18) or variant translocations involving chromosome 18, they should either be separated as a subgroup or included into the group of follicle center lymphomas.

Variant forms of Philadelphia translocation in two patients with chronic myeloid leukemia.

During a 4-year period (December 1990-December 1994), among other diagnoses one hundred cases of chronic myeloid leukemia (CML) were analyzed in our department. We focused our attention on two cases with a variant form of Philadelphia translocation. Cytogenetic and molecular genetic studies were performed to resolve the status of BCR and ABL in the bone marrow or peripheral blood cells of the two CML patients with complex translocations involving chromosomes 3, 9, 22 and 9, 12, 22 respectively. In the first case the presence of Ph chromosome was detected cytogenetically, BCR-ABL translocation was detected by Southern hybridization. In the second case, only the PCR method showed BCR-ABL rearrangement. The second case, with a random variant form of Ph translocation, could be detected using different methods of clinical molecular genetics.

FMS hemizygosity in myeloid dysplasia and acute myeloid leukemia with chromosomal aberration del(5)(q) demonstrated by polymerase chain reaction.

Interstitial deletions of the long arm of chromosome 5 del(5)(q), are recurring aberrations in the myelodysplastic syndrome and acute myeloid leukemia. Several genes located in region (5)(q23-34) have been implicated as being of pathogenic importance. In this study seven samples of six patients with myelodysplastic syndrome and acute myeloid leukemia who have the del(5)(q) aberration were analyzed by polymerase chain reaction (PCR) and Southern blot technique. FMS hemizygosity was demonstrated in all patients. PCR analysis from peripheral blood samples confirmed the observations of this aberration found by semiquantitative Southern blot. PCR-based analysis can be used for primary diagnosis in addition to cytogenetic evaluation and for follow-up in patients with del(5)(q) aberration.

Analysis of peripheral blood progenitor cells demonstrates limitations of minimal residual disease diagnosis in a case of acute promyelocytic leukemia.

Detection of minimal residual disease (MRD) by analysis of the PML-RAR alpha fusion transcript using the RT-PCR method is routinely carried out on peripheral blood and bone marrow of patients with APL (AML, FAB:M3). Therapy aims to achieve repeated negative results in these patients thus confirming clinical complete remission. We report a case of APL in second complete remission in which no leukemic cells had been detected in BM and PB for 20 months, and in which PBPC-pheresis was carried out for future transplantation. In two of five pheresis PML-RAR alpha fusion transcripts were detected. This shows that the residual leukemic population may only reach detection level after enrichment by PBPC-pheresis.