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Biomedical subjects

H Karlic

Publications and source records attributed to H Karlic.

At least 19 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2005

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.

Adaptation, Physiological↗
PubMed2001

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.

Actins↗
PubMed2001

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.

Adolescent↗
PubMed2001

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.

Animals↗
PubMed2000

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.

Adult↗
PubMed1999

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.

Antigens, CD34↗
PubMed1997

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.

Adult↗
PubMed1996

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.

Adult↗
PubMed1996

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.

Acute Disease↗
PubMed1996

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.

Adult↗
PubMed1995

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.

Acute Disease↗
PubMed1995

Early detection of minimal residual disease by reverse transcriptase polymerase chain reaction predicts relapse in acute promyelocytic leukemia.

The PML/RAR alpha fusion RNA can be detected in acute promyelocytic leukemia (APL), cytogenetically characterized by the translocation t(15;17). Our study included ten newly diagnosed patients with APL who were investigated during the course of their diseases using reverse transcription polymerase chain reaction (RT-PCR). At diagnosis, aberrant fragments with a size heterogeneity due to alternative spliced products were detected in all patients, we observed breakpoints within bcr3 (short type) in two patients and bcr1 and 2 breakpoints (long type) in eight patients. Treatment consisted of all-trans retinoic acid (ATRA) in all patients; six patients received simultaneous cytostatic therapy during remission induction. At the time of complete hematological remission (CR), only two patients showed a negative RT-PCR result; eight of the ten patients were still PCR positive when nested primers were used. Subsequently, eight patients received consolidation chemotherapy and became PCR negative. Seven of eight patients are in continuous complete remission (median remission duration: 21 months, range: 11+ -26+ months). One patient of the chemotherapy group became PCR positive after 4 months in complete remission and relapsed after 6 months. The remaining two patients who were treated only with ATRA relapsed, received induction chemotherapy, and are in second and third complete remission, respectively. In conclusion. PCR negativity can be achieved only by chemotherapeutic consolidation; patients treated with ATRA alone remain PCR positive. Relapse is always preceded by a positive PCR result. Surprisingly, also patients without measurable PML/RAR alpha-mRNA in sequential analyses after cytostatic treatment became PCR positive and experienced relapse.

Adult↗
PubMed1994

Absence of N-ras mutations in myeloid and lymphoid blast crisis of chronic myeloid leukemia.

Mutations within N-ras oncogene codons 12, 13, and 61 occur in approximately 25-30% of patients with acute nonlymphocytic leukemia and at a lower frequency (6-20%) in patients with acute lymphocytic leukemia. Moreover, N-ras mutations have been described in patients with chronic myeloid leukemia (CML) in blast crisis but have not been observed during the chronic phase of the disease. In view of the morphological and clinical similarities between acute leukemia and the blast crisis of CML, the question was raised whether the presence of N-ras mutations is associated with the phenotype of acute leukemia. We investigated leukemic cells from 100 patients with CML for the presence of N-ras mutations in the mutational hot spot codons. The cases analyzed included 87 diagnosed with different types of blast crisis and 13 cases in accelerated or chronic phase of the disease. Fragments from N-ras exons I and II containing the codons of interest were amplified by polymerase chain reaction and analyzed for the presence of point mutations by three different technical approaches, including specific oligonucleotide hybridization, direct sequencing, and single-strand conformation polymorphism analysis. N-ras mutations were not detected in any of the CML patients investigated. Only one patient, in whom the initial diagnosis of CML-blast crisis had been revised to chronic myelomonocytic leukemia, displayed an N-ras mutation within codon 13. Our data strongly suggest that N-ras mutations do not play a role in myeloid or lymphoid blast crisis of CML.

Base Sequence↗
PubMed1992

Minor BCR (m-bcr) rearrangements may appear in major BCR (M-bcr)-positive CML cases.

The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from the reciprocal translocation t(9;22) (q34;q11). On DNA level a BCR/ABL rearrangement involving the so-called major BCR (Mbcr) from chromosome 22 has been associated with chronic myeloid leukemia (CML). For Ph+ ALL a site of rearrangements in the 5' part of the BCR (breakpoint cluster region) gene on chromosome 22, the so-called minor bcr region (mbcr) has been described within the first intron in a 10.8 kb region (=bcr2 or m-BCR1). The BB1 probe detects two Eco fragments of 8.5 and/or 11 kb, which may appear as monomorphic or heteromorphic alleles, both covering bcr2. We have analyzed EcoRI restriction polymorphisms within bcr2 in 42 patients with a rearrangement in M-bcr (including 39 Philadelphia chromosome-positive (Ph+) CML patients and 3 ALLs) and in 18 healthy unrelated volunteers. Of the 42 patients tested, 52.4% (22) had the 8.5 kb bcr2 allele, 21.4% (9) had the 11 kb bcr2 allele, and 26.2% (11) had both the 8.5 and the 11 kb allele. In addition to normal allelic polymorphisms in bcr2, rRFs (rearranged bcr2 restriction fragments) were found in bcr2 as shown in 33% (14 of 42) of our patients. By contrast, no rRFs were found in 18 healthy volunteers. Our results indicate, that heterogeneous rearrangements in bcr2 may appear in addition to BCR/ABL rearrangements involving M-bcr in Ph+CML.

Alleles↗
PubMed1990

Structural chromosomal abnormalities in gynecologic malignancies.

Surgical specimens taken from four patients with gynecologic malignancies were cultured, and metaphase chromosomes were prepared after staining with chromamycin-A, distamycin, and DAPI. Four specially selected karyotypes and their structural aberrations are discussed in this study and compared with those (also from carcinomas) previously described in the literature.

Adenocarcinoma↗