Sustaining proliferative signaling is one of the hallmarks of cancer. Granulocyte-colony stimulat... more Sustaining proliferative signaling is one of the hallmarks of cancer. Granulocyte-colony stimulating factor (G-CSF), a hematopoietic growth factor that controls proliferation and differentiation of neural stem cells, may contribute to the development of glioma. In the present study, we evaluated the expression of G-CSF and its receptor (G-CSFR) in various grades of glioma samples and primary cell culture derived from a glioblastoma patient as well as different human glioma cell lines. We showed that G-CSF and G-CSFR were widely expressed by glioma samples, primary glioma cell culture and glioma cell lines. The expression levels of G-CSF and G-CSFR were not significantly different between different grades of glioma. G-CSF promoted proliferation, migration and invasion of glioma cells. Neutralization of G-CSFR with its antibody inhibited growth and metastasis of glioma cells in vitro. We also showed that activation of signal transducer and activator transcription 3 (STAT 3) as well as expression of several of its downstream effectors was regulated by G-CSF. Taken together, the present results suggest that G-CSF contributes to glioma progression that may be linked to glioma genesis and recurrence.
A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among ... more A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length...
Hematopoietic stem cells (HSCs) reside mostly in the bone marrow and are defined by their ability... more Hematopoietic stem cells (HSCs) reside mostly in the bone marrow and are defined by their ability to self-renew and to give rise by proliferation and differentiation to all blood lineages. Despite this strict definition HSCs cannot be unequivocally identified in the hematopoietic cell pool. Despite innumerable studies over the years, which focused on the search of the ideal phenotypic marker to selectively isolate stem cells, most of the known markers still define heterogeneous populations in different stages of commitment. Functional features attributed to stem cells have also been investigated, and among these the use of fluorescent markers which allow tracking of the cell division record of each cell. A second issue, after the initial isolation process, is the expansion ex vivo in order to obtain production of large numbers of homogeneous cell populations for both biological studies and clinical applications. Expansion ex vivo is difficult to modulate and normally occurs only along with commitment and consequent loss of multipotentiality. Moreover expansion obtained ex vivo is significantly reduced to that achievable in vivo. One of the key features of HSCs is a very slow proliferation rate, but when the appropriate stimuli are delivered, the proliferation rate can drastically increase. In normal physiological conditions a strict balance is maintained between the number of cells that maintain the original pool and those that proliferate and differentiate. Numerous data in recent years are providing some clue to elucidate the key steps in this tightly controlled process, but the dynamics that regulate which and how many cells self-renew to maintain the pool, and which proliferate and become committed to give rise to the mature blood elements, are still unclear.
A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among ... more A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor 1 (TGF-1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-1 maintains human CD34 ؉ hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-1 upregulates the human CD34 antigen in the CD34 ؉ cell lines TF-1 and KG-1a, but not in the more differentiated CD34 ؊ cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-1 has been shown to activate a TGF--activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-1 and CD34 overexpression. Our data demonstrate that TGF-1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents. Leukemia (2002) 16, 94-105.
Purpose In the last years, the influence of different genes involved in metabolism of chemotherap... more Purpose In the last years, the influence of different genes involved in metabolism of chemotherapeutic agents has been studied. Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of acute lymphoblastic leukemia (ALL), primary central nervous system lymphoma (PCNSL) and Burkitt's lymphomas (BL). This study aims to evaluate the role of MTHFR C677T and A1298C polymorphisms and G80A reduced folate carrier gene (RFC1) in a cohort of adult patients with lymphoproliferative malignancies submitted to high-dose MTX followed by leucovorin rescue. Methods We performed the analysis of these polymorphisms on genomic DNA with RFLP-PCR. Results Patients carrying MTHFR A1298C variant showed decreased hepatic and hematological toxicity (P = 0.03). Overall survival (OS) and progression-free survival (PFS) between homozygous wild-type and variant patients for the RFC1 G 80 A were significantly different (P = 0.035 and P = 0.02, respectively). A significant correlation between hematological toxicity and age (P = 0.003) was observed. There was no significant influence of MTHFR C677T genotype on toxicity, OS and PFS. Conclusions Leucovorin rescue given after high-dose MTX probably accounts for the lack of influence of C677T polymorphism. To better define a role of RFC1 polymorphism on patients outcome, it would be worthwhile to perform a study on intracellular MTX level and RFC1 substrate binding affinities in different genotypes.
Stem/progenitor cells endowed with in vitro and in vivo haematopoietic activity express the surfa... more Stem/progenitor cells endowed with in vitro and in vivo haematopoietic activity express the surface protein CD34. Transforming growth factor b1 (TGF-b1) is one of the soluble molecules that regulate cell cycle and differentiation of haematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has previously been shown that TGF-b1 maintains human CD34 + haematopoietic progenitors in an undifferentiated state, independently of any cell cycle effect. Here, we have shown that TGF-b1 upregulates the human CD34, an effect that was evident in primary stem/progenitor cells (CD34 + lin -) both at the transcriptional and protein levels, and was not associated with any relevant effect on cell growth. The presence of TGF-b1 influenced differentiation, maintaining primary CD34 + /Linin an undifferentiated state. This effect was associated with Smad activation and with a dramatic decrease in p38 phosphorylation. Moreover, blocking p38 phosphorylation by the SB202190 inhibitor increased CD34 RNA levels but did not enhance CD34 protein expression in CD34 + /Lincells, suggesting that modulation of multiple signalling pathways is necessary to reproduce TGF-b1 effects. These data establish the role that TGF-b1 has in the modulation of the CD34 stem/ progenitor protein and stem/progenitor functions, providing important clues for understanding haematopoietic development and a potential tool for the modulation of human haematopoiesis.
We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as... more We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells.
Genetic polymorphisms affecting methylentetrahydrofolate reductase (MTHFR) activity may influence... more Genetic polymorphisms affecting methylentetrahydrofolate reductase (MTHFR) activity may influence hematological and neurological dysfunction in cobalamin-deficient patients. We studied the prevalence of C677T and A1298C polymorphisms by analyzing genomic DNA in 30 cobalamin-deficient patients. No significant difference was found in 677 and 1298 genotype distribution with respect to hematological parameters, B12 and folate levels, and neurological symptoms. The two MTHFR polymorphisms were not protective against anemia or neurological dysfunction in patients with cobalamin deficiency; however, we found evidence of a significant increase in atrophic gastritis in the 677TT group (P = 0.009) but not for the 1298CC genotype. Based on observations that inadequate cobalamin intake and reduced MTHFR activity might be significant risk factors for gastric cancer, and the increased risk of gastric cancer shown in patients affected by atrophic gastritis, we speculate that concomitant atrophic gastritis and impaired MTHFR function could have a role in the development of gastric cancer.
Sustaining proliferative signaling is one of the hallmarks of cancer. Granulocyte-colony stimulat... more Sustaining proliferative signaling is one of the hallmarks of cancer. Granulocyte-colony stimulating factor (G-CSF), a hematopoietic growth factor that controls proliferation and differentiation of neural stem cells, may contribute to the development of glioma. In the present study, we evaluated the expression of G-CSF and its receptor (G-CSFR) in various grades of glioma samples and primary cell culture derived from a glioblastoma patient as well as different human glioma cell lines. We showed that G-CSF and G-CSFR were widely expressed by glioma samples, primary glioma cell culture and glioma cell lines. The expression levels of G-CSF and G-CSFR were not significantly different between different grades of glioma. G-CSF promoted proliferation, migration and invasion of glioma cells. Neutralization of G-CSFR with its antibody inhibited growth and metastasis of glioma cells in vitro. We also showed that activation of signal transducer and activator transcription 3 (STAT 3) as well as expression of several of its downstream effectors was regulated by G-CSF. Taken together, the present results suggest that G-CSF contributes to glioma progression that may be linked to glioma genesis and recurrence.
A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among ... more A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor beta1 (TGF-beta1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-beta1 maintains human CD34(+) hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-beta1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-beta1 upregulates the human CD34 antigen in the CD34(+) cell lines TF-1 and KG-1a, but not in the more differentiated CD34(-) cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-beta1 did not effect cell growth, but induced transcriptional activation of full-length...
Hematopoietic stem cells (HSCs) reside mostly in the bone marrow and are defined by their ability... more Hematopoietic stem cells (HSCs) reside mostly in the bone marrow and are defined by their ability to self-renew and to give rise by proliferation and differentiation to all blood lineages. Despite this strict definition HSCs cannot be unequivocally identified in the hematopoietic cell pool. Despite innumerable studies over the years, which focused on the search of the ideal phenotypic marker to selectively isolate stem cells, most of the known markers still define heterogeneous populations in different stages of commitment. Functional features attributed to stem cells have also been investigated, and among these the use of fluorescent markers which allow tracking of the cell division record of each cell. A second issue, after the initial isolation process, is the expansion ex vivo in order to obtain production of large numbers of homogeneous cell populations for both biological studies and clinical applications. Expansion ex vivo is difficult to modulate and normally occurs only along with commitment and consequent loss of multipotentiality. Moreover expansion obtained ex vivo is significantly reduced to that achievable in vivo. One of the key features of HSCs is a very slow proliferation rate, but when the appropriate stimuli are delivered, the proliferation rate can drastically increase. In normal physiological conditions a strict balance is maintained between the number of cells that maintain the original pool and those that proliferate and differentiate. Numerous data in recent years are providing some clue to elucidate the key steps in this tightly controlled process, but the dynamics that regulate which and how many cells self-renew to maintain the pool, and which proliferate and become committed to give rise to the mature blood elements, are still unclear.
A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among ... more A number of cytokines modulate self-renewal and differentiation of hematopoietic elements. Among these is transforming growth factor 1 (TGF-1), which regulates cell cycle and differentiation of hematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has been previously shown by us and other authors that TGF-1 maintains human CD34 ؉ hematopoietic progenitors in an undifferentiated state, independently of any cell cycle effects, and that depletion of TGF-1 triggers differentiation accompanied by a decrease in CD34 antigen expression. In the present work, we show that exogenous TGF-1 upregulates the human CD34 antigen in the CD34 ؉ cell lines TF-1 and KG-1a, but not in the more differentiated CD34 ؊ cell lines HL-60 and K-562. We further studied this effect in the pluripotent erythroleukemia cell line TF-1. Here, TGF-1 did not effect cell growth, but induced transcriptional activation of full-length CD34 and prevented differentiation induced by differentiating agents. This effect was associated with nuclear translocation of Smad-2, activation of TAK-1, and with a dramatic decrease in p38 phosphorylation. In other systems TGF-1 has been shown to activate a TGF--activated kinase 1 (TAK1), which in turn, activates p38. The specific inhibitor of p38 phosphorylation, SB202190, also increased CD34 RNA expression, indicating the existence of a link between p-38 inhibition by TGF-1 and CD34 overexpression. Our data demonstrate that TGF-1 transcriptionally activates CD34 and prevents differentiation of TF-1 cells by acting independently through the Smad, TAK1 and p38 pathways, and thus provide important clues for the understanding of hematopoietic development and a potential tool to modify response of hematopoietic cells to mitogens or differentiating agents. Leukemia (2002) 16, 94-105.
Purpose In the last years, the influence of different genes involved in metabolism of chemotherap... more Purpose In the last years, the influence of different genes involved in metabolism of chemotherapeutic agents has been studied. Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of acute lymphoblastic leukemia (ALL), primary central nervous system lymphoma (PCNSL) and Burkitt's lymphomas (BL). This study aims to evaluate the role of MTHFR C677T and A1298C polymorphisms and G80A reduced folate carrier gene (RFC1) in a cohort of adult patients with lymphoproliferative malignancies submitted to high-dose MTX followed by leucovorin rescue. Methods We performed the analysis of these polymorphisms on genomic DNA with RFLP-PCR. Results Patients carrying MTHFR A1298C variant showed decreased hepatic and hematological toxicity (P = 0.03). Overall survival (OS) and progression-free survival (PFS) between homozygous wild-type and variant patients for the RFC1 G 80 A were significantly different (P = 0.035 and P = 0.02, respectively). A significant correlation between hematological toxicity and age (P = 0.003) was observed. There was no significant influence of MTHFR C677T genotype on toxicity, OS and PFS. Conclusions Leucovorin rescue given after high-dose MTX probably accounts for the lack of influence of C677T polymorphism. To better define a role of RFC1 polymorphism on patients outcome, it would be worthwhile to perform a study on intracellular MTX level and RFC1 substrate binding affinities in different genotypes.
Stem/progenitor cells endowed with in vitro and in vivo haematopoietic activity express the surfa... more Stem/progenitor cells endowed with in vitro and in vivo haematopoietic activity express the surface protein CD34. Transforming growth factor b1 (TGF-b1) is one of the soluble molecules that regulate cell cycle and differentiation of haematopoietic cells, but has pleiotropic activities depending on the state of responsiveness of the target cells. It has previously been shown that TGF-b1 maintains human CD34 + haematopoietic progenitors in an undifferentiated state, independently of any cell cycle effect. Here, we have shown that TGF-b1 upregulates the human CD34, an effect that was evident in primary stem/progenitor cells (CD34 + lin -) both at the transcriptional and protein levels, and was not associated with any relevant effect on cell growth. The presence of TGF-b1 influenced differentiation, maintaining primary CD34 + /Linin an undifferentiated state. This effect was associated with Smad activation and with a dramatic decrease in p38 phosphorylation. Moreover, blocking p38 phosphorylation by the SB202190 inhibitor increased CD34 RNA levels but did not enhance CD34 protein expression in CD34 + /Lincells, suggesting that modulation of multiple signalling pathways is necessary to reproduce TGF-b1 effects. These data establish the role that TGF-b1 has in the modulation of the CD34 stem/ progenitor protein and stem/progenitor functions, providing important clues for understanding haematopoietic development and a potential tool for the modulation of human haematopoiesis.
We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as... more We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells.
Genetic polymorphisms affecting methylentetrahydrofolate reductase (MTHFR) activity may influence... more Genetic polymorphisms affecting methylentetrahydrofolate reductase (MTHFR) activity may influence hematological and neurological dysfunction in cobalamin-deficient patients. We studied the prevalence of C677T and A1298C polymorphisms by analyzing genomic DNA in 30 cobalamin-deficient patients. No significant difference was found in 677 and 1298 genotype distribution with respect to hematological parameters, B12 and folate levels, and neurological symptoms. The two MTHFR polymorphisms were not protective against anemia or neurological dysfunction in patients with cobalamin deficiency; however, we found evidence of a significant increase in atrophic gastritis in the 677TT group (P = 0.009) but not for the 1298CC genotype. Based on observations that inadequate cobalamin intake and reduced MTHFR activity might be significant risk factors for gastric cancer, and the increased risk of gastric cancer shown in patients affected by atrophic gastritis, we speculate that concomitant atrophic gastritis and impaired MTHFR function could have a role in the development of gastric cancer.
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