Cer1p functions as a molecular chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae.

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.

Adequate tumour quinidine levels for multidrug resistance modulation can be achieved in vivo.

The multidrug resistance (MDR) phenotype can be reversed in vitro by a number of agents thought to interact with P-glycoprotein (P-gp). Although plasma levels, adequate for MDR modulation, can be achieved with certain modulators, concern has been expressed that tumour levels may be inadequate due to high plasma protein binding. Mice bearing an MDR-positive human tumour xenograft were injected intraperitoneally with quinidine (150 mg/kg). After 2 h the mean plasma quinidine level was 1.9 micrograms/ml (5.1 mumol/l) and the mean tumour quinidine effective in vitro. Three tumour biopsy specimens were obtained from patients who had received oral quinidine prior to surgery. Plasma and tumour levels were similar and were comparable with those measured in mice. This study should dispel fears of inadequate tumour levels of this and other modulators due to high plasma protein binding and encourage future clinical trials of modulators in MDR-positive human tumours.

Circumvention of pleiotropic drug resistance in subcutaneous tumours in vivo with verapamil and clomipramine.

The development of pleiotropic drug resistance (PDR) in vivo in solid tumour models suggests that a similar process may occur in the clinic. A subline of the Ridgway osteogenic sarcoma (ROS)--a murine subcutaneously-growing solid tumour--with moderate resistance (1.5 fold) to actinomycin D was selected by repeated suboptimal treatment with this drug in vivo. This subline (ROS/ADX/G2) showed cross-resistance to vincristine (3.5 fold) and etoposide (over 5.1 fold) but not to doxorubicin. The resistance could in all cases be partly or completely overcome by treatment with non-cytotoxic doses of verapamil or clomipramine. Resistance to actinomycin in this model was associated with lower (up to 3.2 fold) drug accumulation into tumours which could be increased (up to 2.8 fold) by treatment with 25 micrograms/g verapamil. These data support clinical trials of the use of membrane-active agents to overcome PDR.

Pharmacokinetics and efficacy of 131I-meta-iodobenzylguanidine in two neuroblastoma xenografts.

The pharmacokinetics, biodistribution and efficacy of the radiopharmaceutical 131I-meta-iodobenzylguanidine (131I-mIBG) were determined in murine xenografts of two human neuroblastoma cell lines, SK-N-SH and SK-N-BE(2c). These lines have similar capacities in vitro for active uptake of 131I-mIBG, but different radiobiological characteristics. Groups of four mice were killed after injection of 131I-mIBG, and retained radioactivity in the tumour and normal tissues was measured at 8, 16, 24 and 48 h. Within each type there was heterogeneity of tumour uptake, although average values were similar for both. The per cent injected dose per gram of tumour retained at 24 h was (mean and 95% confidence interval) 0.95 (0.67-1.23) for SK-N-SH and 0.76 (0.47-1.05) for SK-N-BE(2c). The growth of tumours in groups of seven animals following injection of 35, 70 or 105 MBq 131I-mIBG was compared with that of controls. The specific regrowth delay (median and 95% confidence intervals) caused by 105 MBq 131I-mIBG was 4.2 (0.9-5.9) in SK-N-SH and 5.6 (0-11.3) in SK-N-BE(2c) bearing mice. SK-N-BE(2c) xenografts were significantly more sensitive to external beam irradiation than SK-N-SH xenografts.

Cer1p, a novel Hsp70-related protein required for posttranslational endoplasmic reticulum translocation in yeast.

Proteins enter the secretory pathway by translocation across the endoplasmic reticulum (ER) membrane. In Saccharomyces cerevisiae, import of proteins into the ER occurs both cotranslationally and posttranslationally. Presumably, the cotranslational targeting to the ER membrane is directed by the signal recognition particle, as demonstrated in other eukaryotic systems. The deletion of a gene, called CER1, inhibits the translocation of proteins that enter the ER posttranslationally, but not those that enter cotranslationally. This translocation defect is more pronounced at lower temperatures. A strain possessing a null mutation of CER1 in combination with a kar2 temperature-sensitive mutation displays synthetic growth defects, whereas overexpression of the ER DnaJ homolog Scj1p suppresses the translocation defect in cer1Delta strains. CER1 is predicted to encode a 100-kDa polypeptide, residing in the ER lumen that is related to the hsp70 family of molecular chaperones.