Nuclear distribution of porphobilinogen deaminase (PBGD) in glioma cells: a regulatory role in cancer transformation?

Abstract

Recently, considerable interest has been directed to red-fluorescence photodiagnosis of brain and other tumours during surgery using the protoporphyrin IX natural precursor, 5-aminolaevulinic acid. In the present study we focused on the role of the rate-limiting enzyme porphobilinogen deaminase in glioma C6 cell activity, differentiation and sub-cellular distribution. Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using the housekeepingporphobilinogen deaminase plasmid, induced a G1 cell cycle attenuation accompanied by increases in enzyme activity and c6 differentiation toward astrocytes. Visualisation of subcellular localisation of the porphobilinogen deaminase using the independent techniques of fluorescence immuno-staining with specific anti-human porphobilinogen deaminase antibodies and cellular expression of porphobilinogen deaminase fused to green fluorescent protein, revealed (unexpectedly) a major fraction of porphobilinogen deaminase in the nucleus and only a minor fraction in the cytoplasm. Both C and N terminals of porphobilinogen deaminase fused to green fluorescent protein revealed a major fraction of the newly synthesized fused porphobilinogen deaminase in the nucleus. Furthermore, newborn rat brain cells grown in a primary culture showed the same localisation pattern of porphobilinogen deaminase in the nuclei. Stimulation of C6 glioma cell differentiation by butyrate induced a marked decrease in porphobilinogen deaminase both in the nucleus and in the cytoplasm as determined by Western blotting and fluorescence immuno-localisation. These findings suggest a possible dual role for housekeeping porphobilinogen deaminase in fast dividing glioma cells, one related to the porphyrin synthesis pathway and another coupled to nuclear function, which might be linked to tumorigenesis.

Differentiation-dependent photodynamic therapy regulated by porphobilinogen deaminase in B16 melanoma

“…We have already shown that the major fraction of PBGD in C6 glioma cells was found in the nucleus (Greenbaum et al, 2002). Figure 6 reveals the subcellular localisation of PBGD in B16 melanoma cells.…”

“…All the coding regions of the fragments were amplified from pHK-PBGD (pLY-81) (Greenbaum et al, 2002) using different primers for each fragment. The PCR products were cut by the restriction enzymes BglII and EcoRI, and were ligated to pEGFP-N1 (Clontech, Palo Alto, CA, USA) in frame.…”

“…We have recently shown that a major fraction of PBGD in C6 glioma cells is localised in the nucleus, in addition to the cytosolic fraction active in PpIX synthesis (Greenbaum et al, 2002). Furthermore, overexpression of the human housekeeping PBGD has been found to induce C6 differentiation toward astrocytes.…”

A porphobilinogen deaminase (PBGD) Ran-binding protein interaction is implicated in nuclear trafficking of PBGD in differentiating glioma cells

“…We have shown that the PBGD, including the nuclear fraction, is downregulated during butyrate-induced cell differentiation of glioma (Greenbaum et al, 2002) and melanoma cells (not yet published), which points to a possible nuclear function of PBGD. Butyrate-dependent induction leads to histone hyperacetylation via inhibition of histone deacetylase (ADAC), inhibition of histone phosphorylation, and DNA methylation (Archer and Hodin, 1999).…”

“…Recently, we showed that PBGD is imported into the nucleus of glioma C6 cells in addition to the fraction, that remains in the cytoplasm (Greenbaum et al, 2002). Nuclear import of many proteins was shown to be dependent on a specific import mechanism via RanGTPase and on the cytosolic or nuclear localization of this enzyme (Yoneda, 2000;Quimby and Corbett, 2001).…”