The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cel... more The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cell membrane proteins. The structure and functional characteristics of these molecules indicate that they may play important roles in transmembrane signaling in different cells. Recently, it was reported that cross-linking of CD53 on human B cells led to an increase in cytoplasmic calcium fluxes. In the present study, we wished to further explore the possible role of CD53 in functional B cell responses. Cross-linking of CD53 with the use of the mAb MEM-53 and a polyclonal sheep anti-mouse Ig promoted activation of resting B cells into the G1 phase of the cell cycle as judged by increased expression of the early activation Ag CD69, increases in cellular volume, RNA synthesis, and c-myc protein levels, and enhanced binding of 7-aminoactinomycin D. In contrast, MEM-53 alone had no detectable effects. Cross-linking of anti-CD53 induced negligible S phase entry in the absence of other stimuli. However, cytokines, in particular IL-2 and IL-4, potentiated the DNA synthesis induced by cross-linking of CD53.
The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cult... more The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cultured in monolayer, V79 Chinese hamster fibroblasts and WiDr human colon adenocarcinoma cells. The cells were incubated with 5-aminolaevulinic acid (5-ALA) as a precursor for the endogenously synthesised protoporphyrin IX, which was activated by light. Free DNA ends, owing to internucleosomal DNA cleavage in apoptotic cells, were stained specifically with a fluorescent dye in the terminal deoxynucleotidyl transferase (TdT) assay. The free DNA ends were measured by flow cytometry and the fractions of apoptotic cells determined. Total cell death was measured in a cell survival assay to determine the necrotic fraction after subtraction of the apoptotic fraction. V79 cells did undergo apoptosis while WiDr cells were killed only through necrosis. With time, the apoptotic fraction of V79 cells increased until a maximum was reached about 3-4 h after ALA-PDT treatment. For increasing ALA-PDT doses, a maximal apoptotic fraction 75-85% of the cells was measured at about 85% of total cell death. The flow cytometric assay of apoptosis was confirmed by the typical ladder of oligonucleosomal DNA fragments obtained from agarose gel electrophoresis, by fluorescence micrographs visualising the induced free DNA ends and by electron micrographs showing the typical morphology of apoptotic cells.
A solid-phase differential display method was designed to analyze differential gene expression in... more A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the procedure simplified the purification steps, limited sample loss and enabled rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34 + CD38 -). The majority of the differentially expressed fragments represented previously uncharacterized sequences.
PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. ... more PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. The molecular mechanisms and particularly the cellular phenotypes resulting in synthetic lethality are not well understood and varying clinical responses have been observed. We have investigated the dose- and time-dependency of cell growth, cell death and cell cycle traverse of 4 malignant lymphocyte cell lines treated with the PARP inhibitor Olaparib. PARP inhibition induced a severe growth inhibition in this cell line panel and increased the levels of phosphorylated H2AX-associated DNA damage in S phase. Repair of the remaining replication related damage caused a G2 phase delay before entry into mitosis. The G2 delay, and the growth inhibition, was more pronounced in the absence of functional ATM. Further, Olaparib treated Reh and Granta-519 cells died by apoptosis, while U698 and JVM-2 cells proceeded through mitosis with aberrant chromosomes, skipped cytokinesis, and eventually died ...
In contrast with the extensively characterized effects of transforming growth factor-p (TGF-p) on... more In contrast with the extensively characterized effects of transforming growth factor-p (TGF-p) on proliferation and differentiation of hematopoietic progenitors, little is known about the effects of TGF-p on viability of normal hematopoi- etic progenitors. In the present report, we demonstrate that TGF-p potently counteracts hematopoietic growth factor (HGF)-induced survival of individually cultured primitive Lin- -Sea-l+ bone marrow progenitors. Specifically, 74%
Elevated cyclic AMP levels induce a rapid block in the mid-G1 phase of the cell cycle in B-lympho... more Elevated cyclic AMP levels induce a rapid block in the mid-G1 phase of the cell cycle in B-lymphoid Reh cells, accompanied by a transient block in G2. The retinoblastoma (Rb) gene product has been implicated as a key regulator of eukaryotic cell growth. The Rb protein enforces its growth suppressive effect in early G1, where it is underphosphorylated and firmly bound in the nucleus. A possible link between the cyclic AMP-mediated growth arrest and regulation ofRb protein phosphorylation was explored mid-G1.
It is widely accepted that the initiation mass of Escherichia coli is constant and independent of... more It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We have used flow cytometry to measure the initiation mass of E.coli K-12 cells as a function of growth rate. The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution. The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass. The initiation mass was found to increase monotonicaily with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour. We conclude that the initiation mass is dependent on growth rate. This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of replication.
We, therefore, believe that p27 and not pRb is the mediator of this oxygen-dependent checkpoint i... more We, therefore, believe that p27 and not pRb is the mediator of this oxygen-dependent checkpoint in late G 1 . Our results also suggest that p27 regulates the restart of cell cycle progression of these arrested cells after reoxygenation.
Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources u... more Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G 1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G 1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.
Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. Howev... more Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed "G2H" compartment), to cells that there were almost negative and had about 2 foci (termed "G2L" compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2-3 h delay in G2L.
The induction of apoptosis from different intracellular sites was studied by exposing V79 Chinese... more The induction of apoptosis from different intracellular sites was studied by exposing V79 Chinese hamster fibroblasts to photodynamic therapy (PDT) with various porphyrins and light. The effects of two lipophilic, intracellular membrane-localized porphyrins, tetra(3-hydroxyphenyl)porphyrin (3THPP) and Photofrin, were compared with that of two sulphonated meso-tetraphenylporphines (TPPS 2a and TPPS 4 ), which are taken up into lysosomes by endocytosis. Apoptotic fractions induced by the various dyes and light were quantified by flow cytometry using the terminal deoxynucleotidyl transferase (TdT) assay. Cell fragmentation was measured in parallel, while the nuclear morphology of apoptotic cells was studied by fluorescence microscopy. Different kinetics were found for the induction of DNA strand breaks characteristic of apoptotic cells. PDT-induced damage to membranes resulted in an increasing number of apoptotic cells for about 12 h after PDT. After damage to lysosomes, apoptotic cells were not detected until more than 12 h after PDT. Furthermore, apoptotic bodies were not observed after PDT-induced damage to intracellular membranes, whereas apoptosis induced from lysosomal sites was characterized by extensive cell fragmentation. Cell fragmentation occurred in combination with or in the absence of nuclear fragmentation. The results support the idea that the degradation phase of apoptosis can consist of a sequence of independent steps rather than a common final pathway.
Passage through the Retinoblastoma protein (RB1)dependent restriction point and the loading of mi... more Passage through the Retinoblastoma protein (RB1)dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyperphosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells. by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from analyzed by flow cytometry for MCMs and RB1 as in Figure 1B. Lines in the density scatter plots (top and middle) indicate the regions defining positive and negative cells for either MCM or RB1. Gates for G1-cells are indicated in the DNA histograms (bottom). (B) Scatter plots of MCM versus RB1 for the gated G1 populations in (A). (C) Quantification of the percentages of G1 cells in each quadrant from scatter plots as in (B). Mean values from five experiments are shown for BJ (black bars) and U2OS (gray bars) cells. Error bars represent standard error of mean. by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from Nucleic Acids Research, 2015 9 Figure 5. U2OS cells released from Nocodazole block can load MCMs before RB1 hyper-phosphorylation. (A) U2OS cells synchronized by release from Nocodazole-arrest were analyzed by flow cytometry for MCMs and RB1 as in Figure 1B. The time points (2-12 h) indicate times after release from the Nocodazole-arrest. Lines in the density scatter plots (top and middle) indicate the regions defining positive and negative cells for either MCM or RB1. Gates for G1-cells are indicated in the DNA histograms (bottom). (B) Scatter plots of MCM versus RB1 for the gated G1 populations in (A). (C) Column charts showing the percentages of MCM-positive cells (quadrants Q1 and Q2) in the scatter plots shown in (B). Mean values from three independent experiments are shown. Error bars represent standard error of the mean. (D) Immunoblot analysis as in Figure 2A of unextracted and extracted U2OS cells. Cells were synchronized and released as in (A). The time points indicate time after release from Nocodazole-arrest. by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from
Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causat... more Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.
Six new human melanoma xenograft lines differing considerably in biological properties were estab... more Six new human melanoma xenograft lines differing considerably in biological properties were established in athymic nude mice and characterized with respect to growth in vivo. Mean volume-doubling time ranged from 1.8 to 17.7 days, mean necrotic fraction from 7 to 39%, and mean fraction of cells in S from 11 to 31% at a tumor volume of 500 mm3. DNA-index and median chromosome number were in the ranges 1.15-1.76 and 46-80, respectively. The lines were all highly malignant by histopathological criteria, but showed significant differences with respect to cell type, cellular pleomorphism, nuclear atypia, nucleolar prominence, and mitotic activity. Several biological characteristics of the donor patients' melanomas were retained in the xenograft lines. All lines showed a human karyotype and the histological appearance was very similar to that of the source tumor material. The distribution of cells in the cell cycle was comparable to that reported for melanomas in man. The six melanoma xenograft lines constitute a tumor panel that shows great promise for future studies of the biology and treatment sensitivity of malignant melanoma.
A low quantity of mitochondrial DNA (mtDNA) is a risk factor in a variety of tumor types. However... more A low quantity of mitochondrial DNA (mtDNA) is a risk factor in a variety of tumor types. However, it is unclear how mtDNA reduction influences tumor behavior. mtDNA-deficient ovarian cancer cells were established by ethidium bromide (EtBr) treatment with additive combination of pyruvate and uridine. The mtDNA-deficient cells had a low growth and colony-forming efficiency compared to the control cells. RNA sequencing revealed down-regulation of mitochondrion-related genes and up-regulation of genes related to cell proliferation and anti-apoptosis. The expression of genes involved in cancer metastasis, proliferation, angiogenesis, drug resistance and cancer cell stemness were also up-regulated. Intriguingly, cancer stem cell markers CD90 and CD117 were both up-regulated by EtBr dose-dependently in both cell lines. MtDNA deficiency may induce ovarian cancer stem cell-like properties through different ways in vitro, therefore contributing to different tumor behaviors.
Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma pro... more Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of...
p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the co... more p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the corresponding normal mucosas, and correlated with tumor DNA ploidy as measured by flow cytometry. p53 protein was detected in 35% (12/34) of the tumors; the normal mucosas were negative. Fifty-five percent (12/22) of the tumors examined for mutations within the four hotspots (exons 5-8) of the p53 gene had point mutations. p53 expression correlated significantly with the presence of p53 gene mutations; 67% (8/12) of the tumors with mutations expressed p53, whereas only one of 10 tumors where no mutations were detected expressed the protein (p=0.01). Four tumors with p53 gene mutations did not express p53. Fifty-nine percent (20/34) of the tumors were aneuploid. p53 expression correlated significantly with aneuploidy; a total of 55% (11/20) of the aneuploid tumors were positive for p53 compared to 7% (1/14) of the diploid tumors (p=0.009). All of the 11 highly aneuploid tumors (1.31 less-th...
A better understanding of the development of metastatic disease and the identification of molecul... more A better understanding of the development of metastatic disease and the identification of molecular markers for cancer spread would be useful for the design of improved treatment strategies. This study was conducted to identify gene expressions associated with metastatic phenotypes of locally advanced cervical carcinomas and investigate whether gains or losses of these genes could play a role in regulation of the transcripts. Gene expressions and copy number changes were determined in primary tumors from 29 patients with and 19 without diagnosed lymph node metastases by use of cDNA and genomic microarray techniques, respectively. Thirty-one genes that differed in expression between the node positive and negative tumors were identified. Expressions of eight of these genes (MRPL11, CKS2, PDK2, MRPS23, MSN, TBX3, KLF3, LSM3) correlated with progression free survival in univariate analysis and were therefore more strongly associated with metastatic phenotypes than the others. Immunohist...
The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cel... more The CD53 pan-leukocyte glycoprotein is a member of the recently described tetraspan family of cell membrane proteins. The structure and functional characteristics of these molecules indicate that they may play important roles in transmembrane signaling in different cells. Recently, it was reported that cross-linking of CD53 on human B cells led to an increase in cytoplasmic calcium fluxes. In the present study, we wished to further explore the possible role of CD53 in functional B cell responses. Cross-linking of CD53 with the use of the mAb MEM-53 and a polyclonal sheep anti-mouse Ig promoted activation of resting B cells into the G1 phase of the cell cycle as judged by increased expression of the early activation Ag CD69, increases in cellular volume, RNA synthesis, and c-myc protein levels, and enhanced binding of 7-aminoactinomycin D. In contrast, MEM-53 alone had no detectable effects. Cross-linking of anti-CD53 induced negligible S phase entry in the absence of other stimuli. However, cytokines, in particular IL-2 and IL-4, potentiated the DNA synthesis induced by cross-linking of CD53.
The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cult... more The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cultured in monolayer, V79 Chinese hamster fibroblasts and WiDr human colon adenocarcinoma cells. The cells were incubated with 5-aminolaevulinic acid (5-ALA) as a precursor for the endogenously synthesised protoporphyrin IX, which was activated by light. Free DNA ends, owing to internucleosomal DNA cleavage in apoptotic cells, were stained specifically with a fluorescent dye in the terminal deoxynucleotidyl transferase (TdT) assay. The free DNA ends were measured by flow cytometry and the fractions of apoptotic cells determined. Total cell death was measured in a cell survival assay to determine the necrotic fraction after subtraction of the apoptotic fraction. V79 cells did undergo apoptosis while WiDr cells were killed only through necrosis. With time, the apoptotic fraction of V79 cells increased until a maximum was reached about 3-4 h after ALA-PDT treatment. For increasing ALA-PDT doses, a maximal apoptotic fraction 75-85% of the cells was measured at about 85% of total cell death. The flow cytometric assay of apoptosis was confirmed by the typical ladder of oligonucleosomal DNA fragments obtained from agarose gel electrophoresis, by fluorescence micrographs visualising the induced free DNA ends and by electron micrographs showing the typical morphology of apoptotic cells.
A solid-phase differential display method was designed to analyze differential gene expression in... more A solid-phase differential display method was designed to analyze differential gene expression in samples with low amounts of mRNA. The principle was based on using a biotinylated probe to capture the mRNA and priming both the first-strand synthesis and the subsequent polymerase chain reaction step. Coupling the mRNA to a solid phase during the procedure simplified the purification steps, limited sample loss and enabled rapid handling of mRNA. DNA contamination was also minimized when the mRNA was bound to a solid phase. Optimization of the differential display method was achieved by analyzing both the enzymatic conditions and the required cell amounts. The approach was used for the characterization of genes expressed in the most immature hematopoietic progenitor cells (CD34 + CD38 -). The majority of the differentially expressed fragments represented previously uncharacterized sequences.
PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. ... more PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. The molecular mechanisms and particularly the cellular phenotypes resulting in synthetic lethality are not well understood and varying clinical responses have been observed. We have investigated the dose- and time-dependency of cell growth, cell death and cell cycle traverse of 4 malignant lymphocyte cell lines treated with the PARP inhibitor Olaparib. PARP inhibition induced a severe growth inhibition in this cell line panel and increased the levels of phosphorylated H2AX-associated DNA damage in S phase. Repair of the remaining replication related damage caused a G2 phase delay before entry into mitosis. The G2 delay, and the growth inhibition, was more pronounced in the absence of functional ATM. Further, Olaparib treated Reh and Granta-519 cells died by apoptosis, while U698 and JVM-2 cells proceeded through mitosis with aberrant chromosomes, skipped cytokinesis, and eventually died ...
In contrast with the extensively characterized effects of transforming growth factor-p (TGF-p) on... more In contrast with the extensively characterized effects of transforming growth factor-p (TGF-p) on proliferation and differentiation of hematopoietic progenitors, little is known about the effects of TGF-p on viability of normal hematopoi- etic progenitors. In the present report, we demonstrate that TGF-p potently counteracts hematopoietic growth factor (HGF)-induced survival of individually cultured primitive Lin- -Sea-l+ bone marrow progenitors. Specifically, 74%
Elevated cyclic AMP levels induce a rapid block in the mid-G1 phase of the cell cycle in B-lympho... more Elevated cyclic AMP levels induce a rapid block in the mid-G1 phase of the cell cycle in B-lymphoid Reh cells, accompanied by a transient block in G2. The retinoblastoma (Rb) gene product has been implicated as a key regulator of eukaryotic cell growth. The Rb protein enforces its growth suppressive effect in early G1, where it is underphosphorylated and firmly bound in the nucleus. A possible link between the cyclic AMP-mediated growth arrest and regulation ofRb protein phosphorylation was explored mid-G1.
It is widely accepted that the initiation mass of Escherichia coli is constant and independent of... more It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We have used flow cytometry to measure the initiation mass of E.coli K-12 cells as a function of growth rate. The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution. The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass. The initiation mass was found to increase monotonicaily with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour. We conclude that the initiation mass is dependent on growth rate. This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of replication.
We, therefore, believe that p27 and not pRb is the mediator of this oxygen-dependent checkpoint i... more We, therefore, believe that p27 and not pRb is the mediator of this oxygen-dependent checkpoint in late G 1 . Our results also suggest that p27 regulates the restart of cell cycle progression of these arrested cells after reoxygenation.
Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources u... more Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G 1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G 1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.
Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. Howev... more Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed "G2H" compartment), to cells that there were almost negative and had about 2 foci (termed "G2L" compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2-3 h delay in G2L.
The induction of apoptosis from different intracellular sites was studied by exposing V79 Chinese... more The induction of apoptosis from different intracellular sites was studied by exposing V79 Chinese hamster fibroblasts to photodynamic therapy (PDT) with various porphyrins and light. The effects of two lipophilic, intracellular membrane-localized porphyrins, tetra(3-hydroxyphenyl)porphyrin (3THPP) and Photofrin, were compared with that of two sulphonated meso-tetraphenylporphines (TPPS 2a and TPPS 4 ), which are taken up into lysosomes by endocytosis. Apoptotic fractions induced by the various dyes and light were quantified by flow cytometry using the terminal deoxynucleotidyl transferase (TdT) assay. Cell fragmentation was measured in parallel, while the nuclear morphology of apoptotic cells was studied by fluorescence microscopy. Different kinetics were found for the induction of DNA strand breaks characteristic of apoptotic cells. PDT-induced damage to membranes resulted in an increasing number of apoptotic cells for about 12 h after PDT. After damage to lysosomes, apoptotic cells were not detected until more than 12 h after PDT. Furthermore, apoptotic bodies were not observed after PDT-induced damage to intracellular membranes, whereas apoptosis induced from lysosomal sites was characterized by extensive cell fragmentation. Cell fragmentation occurred in combination with or in the absence of nuclear fragmentation. The results support the idea that the degradation phase of apoptosis can consist of a sequence of independent steps rather than a common final pathway.
Passage through the Retinoblastoma protein (RB1)dependent restriction point and the loading of mi... more Passage through the Retinoblastoma protein (RB1)dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyperphosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells. by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from analyzed by flow cytometry for MCMs and RB1 as in Figure 1B. Lines in the density scatter plots (top and middle) indicate the regions defining positive and negative cells for either MCM or RB1. Gates for G1-cells are indicated in the DNA histograms (bottom). (B) Scatter plots of MCM versus RB1 for the gated G1 populations in (A). (C) Quantification of the percentages of G1 cells in each quadrant from scatter plots as in (B). Mean values from five experiments are shown for BJ (black bars) and U2OS (gray bars) cells. Error bars represent standard error of mean. by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from Nucleic Acids Research, 2015 9 Figure 5. U2OS cells released from Nocodazole block can load MCMs before RB1 hyper-phosphorylation. (A) U2OS cells synchronized by release from Nocodazole-arrest were analyzed by flow cytometry for MCMs and RB1 as in Figure 1B. The time points (2-12 h) indicate times after release from the Nocodazole-arrest. Lines in the density scatter plots (top and middle) indicate the regions defining positive and negative cells for either MCM or RB1. Gates for G1-cells are indicated in the DNA histograms (bottom). (B) Scatter plots of MCM versus RB1 for the gated G1 populations in (A). (C) Column charts showing the percentages of MCM-positive cells (quadrants Q1 and Q2) in the scatter plots shown in (B). Mean values from three independent experiments are shown. Error bars represent standard error of the mean. (D) Immunoblot analysis as in Figure 2A of unextracted and extracted U2OS cells. Cells were synchronized and released as in (A). The time points indicate time after release from Nocodazole-arrest. by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from by guest on August 11, 2015 http://nar.oxfordjournals.org/ Downloaded from
Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causat... more Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.
Six new human melanoma xenograft lines differing considerably in biological properties were estab... more Six new human melanoma xenograft lines differing considerably in biological properties were established in athymic nude mice and characterized with respect to growth in vivo. Mean volume-doubling time ranged from 1.8 to 17.7 days, mean necrotic fraction from 7 to 39%, and mean fraction of cells in S from 11 to 31% at a tumor volume of 500 mm3. DNA-index and median chromosome number were in the ranges 1.15-1.76 and 46-80, respectively. The lines were all highly malignant by histopathological criteria, but showed significant differences with respect to cell type, cellular pleomorphism, nuclear atypia, nucleolar prominence, and mitotic activity. Several biological characteristics of the donor patients' melanomas were retained in the xenograft lines. All lines showed a human karyotype and the histological appearance was very similar to that of the source tumor material. The distribution of cells in the cell cycle was comparable to that reported for melanomas in man. The six melanoma xenograft lines constitute a tumor panel that shows great promise for future studies of the biology and treatment sensitivity of malignant melanoma.
A low quantity of mitochondrial DNA (mtDNA) is a risk factor in a variety of tumor types. However... more A low quantity of mitochondrial DNA (mtDNA) is a risk factor in a variety of tumor types. However, it is unclear how mtDNA reduction influences tumor behavior. mtDNA-deficient ovarian cancer cells were established by ethidium bromide (EtBr) treatment with additive combination of pyruvate and uridine. The mtDNA-deficient cells had a low growth and colony-forming efficiency compared to the control cells. RNA sequencing revealed down-regulation of mitochondrion-related genes and up-regulation of genes related to cell proliferation and anti-apoptosis. The expression of genes involved in cancer metastasis, proliferation, angiogenesis, drug resistance and cancer cell stemness were also up-regulated. Intriguingly, cancer stem cell markers CD90 and CD117 were both up-regulated by EtBr dose-dependently in both cell lines. MtDNA deficiency may induce ovarian cancer stem cell-like properties through different ways in vitro, therefore contributing to different tumor behaviors.
Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma pro... more Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of...
p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the co... more p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the corresponding normal mucosas, and correlated with tumor DNA ploidy as measured by flow cytometry. p53 protein was detected in 35% (12/34) of the tumors; the normal mucosas were negative. Fifty-five percent (12/22) of the tumors examined for mutations within the four hotspots (exons 5-8) of the p53 gene had point mutations. p53 expression correlated significantly with the presence of p53 gene mutations; 67% (8/12) of the tumors with mutations expressed p53, whereas only one of 10 tumors where no mutations were detected expressed the protein (p=0.01). Four tumors with p53 gene mutations did not express p53. Fifty-nine percent (20/34) of the tumors were aneuploid. p53 expression correlated significantly with aneuploidy; a total of 55% (11/20) of the aneuploid tumors were positive for p53 compared to 7% (1/14) of the diploid tumors (p=0.009). All of the 11 highly aneuploid tumors (1.31 less-th...
A better understanding of the development of metastatic disease and the identification of molecul... more A better understanding of the development of metastatic disease and the identification of molecular markers for cancer spread would be useful for the design of improved treatment strategies. This study was conducted to identify gene expressions associated with metastatic phenotypes of locally advanced cervical carcinomas and investigate whether gains or losses of these genes could play a role in regulation of the transcripts. Gene expressions and copy number changes were determined in primary tumors from 29 patients with and 19 without diagnosed lymph node metastases by use of cDNA and genomic microarray techniques, respectively. Thirty-one genes that differed in expression between the node positive and negative tumors were identified. Expressions of eight of these genes (MRPL11, CKS2, PDK2, MRPS23, MSN, TBX3, KLF3, LSM3) correlated with progression free survival in univariate analysis and were therefore more strongly associated with metastatic phenotypes than the others. Immunohist...
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