Flow cytometric analysis of in vivo polyamine deprivation in Lewis lung carcinoma (3LL) cells using the monoclonal antibody SPM8‐2

Delcros, Jean‐Guy; Lœuillet, Laurence; Chamaillard, L; Royou, Anne; Bouillé, Nathalie; Seiler, Nikolaus; Moulinoux, Jacques‐Philippe

doi:10.1002/(sici)1097-0320(19970301)27:3<255::aid-cyto7>3.3.co;2-q

Cited by 4 publications

(7 citation statements)

References 7 publications

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“…We therefore examined T24 cells for co-localization of Gpc-1 and spermine by confocal immunofluorescence microscopy using anti-Gpc-1 polyclonal antibody and mAb Spm8-2, which recognizes free spermine and spermidine (26). Polyamines bound to DNA or RNA or to HS chains in recycling Gpc-1 should not be accessible to the antibody and thus should be undetectable.…”

Section: Involvement Of Gpc-1 In Sperminementioning

confidence: 99%

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Belting

Mani

Jönsson

et al. 2003

Journal of Biological Chemistry

150

113

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Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in Nunsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.

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Section: Involvement Of Gpc-1 In Sperminementioning

confidence: 99%

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Belting

Mani

Jönsson

et al. 2003

Journal of Biological Chemistry

150

113

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“…Indirect immunofluorescence was performed as described previously (Anderson et al, 1997), using the H3 Ab, H3P Ab, or H11-4 clone (Boehringer Mannheim) at a dilution of 1:400 in KB for 1 h at ambient temperature. Indirect immunofluorescence using mAb SPM8-2 was performed as in Delcros et al (1997). Cells were photographed on Kodak Tmax 400 film using a Zeiss axiophot microscope.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

“…Further evidence for the sensitivity of chromosomal association of polyamines to staurosporine was obtained by indirect immunofluorescence with mAb SPM8-2 that recognizes spermine and spermidine (Delcros et al, 1997). Polyamines are small and present in compartments unreachable by antibodies.…”

Section: Association Of Spermidine and Spermine With Chromosomes Is Controlled By A Staurosporine-sensitive Kinasementioning

confidence: 99%

Phosphorylation-induced Rearrangement of the Histone H3 NH2-terminal Domain during Mitotic Chromosome Condensation

Sauvé

Anderson

Ray

et al. 1999

The Journal of Cell Biology

127

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The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.

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“…Cellular localisation of these species should enable a more precise study of their molecular functions. For this purpose antipolyamines antibodies may be useful tools [2,3].The molecular structure of the binding site of antipolyamine antibodies has not been defined yet. The monoclonal antibody SPM8-2 raised against spermine has been characterized in our laboratory [4,5].…”

mentioning

confidence: 99%

“…Cellular localisation of these species should enable a more precise study of their molecular functions. For this purpose antipolyamines antibodies may be useful tools [2,3].…”

mentioning

confidence: 99%

79 Molecular analyses of the combining site of the anti-spermine monoclonal antibody Spm8-2

Bouillé

Johnston

Debroise

et al. 1998

Biochemical Society Transactions

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Polyamines are ubiquitous low molecular weight organic cations which are essential for cell proliferation and differentiation [l].Their functions are still not fully understood. Cellular localisation of these species should enable a more precise study of their molecular functions. For this purpose antipolyamines antibodies may be useful tools [2,3].The molecular structure of the binding site of antipolyamine antibodies has not been defined yet. The monoclonal antibody SPM8-2 raised against spermine has been characterized in our laboratory [4,5]. Its amino acid sequence was deduced from the nucleotide sequence of the cDNA and molecular modeling studies using the programmes AbM (Oxford Molecular) and Modeller and Insight I1 97 (MSI) were performed in an attempt to identify residues involved in the antibody spermine interaction. Computer generated models of the antibody binding site show a positively charged cleft containing acidic and aromatic residues, predominantly tyrosine and aspartic acid residues from the heavy chain CDR,s. These amino acids probably interact with the terminal amino groups of spermine by electrostatic interactions andor hydrogen bonding. It has been shown experimentally that the strength of the antibody spermine interaction, as measured by Elisa, is strongly dependent on ionic strength and pH. This supports the hypothesis that negatively charged acidic residues in the antibody binding site combine with the positively charged amine groups of the polyamine. The affinity of the antibody for spermine decreased dramatically when the NaCl concentration in the ELISA buffer was increased from 0.14 to 0.75M as well as when the pH was decreased below 6 or increased over 8 (Table 1). Table 1 : Effect of ionic strength and pH on the relative affinity (Kax) of Spm8-2 for spermine NaCl (M) 0.15 0.25 0.50 0.75 1

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Flow cytometric analysis of in vivo polyamine deprivation in Lewis lung carcinoma (3LL) cells using the monoclonal antibody SPM8‐2

Cited by 4 publications

References 7 publications

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells

Phosphorylation-induced Rearrangement of the Histone H3 NH2-terminal Domain during Mitotic Chromosome Condensation

79 Molecular analyses of the combining site of the anti-spermine monoclonal antibody Spm8-2

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