Assessment of different transfection parameters in efficiency optimization.

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 +/- 2.28%; p = 0.001) followed by M199 (24 +/- 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 +/- 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 +/- 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 +/- 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 +/- 1.54% to 27 +/- 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.

Assessment of Different Transfection Parameters in Efficiency Optimization.

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 ± 2.28%; p = 0.001) followed by M199 (24 ± 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 ± 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 ± 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 ± 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 ± 1.54% to 27 ± 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.

In vitro senescence occurring in normal human endothelial cells can be rescued by ectopic telomerase activity.

Telomerase activation is a means to delay in vitro replicative senescence in human cells via telomere maintainence; however, this enzymatic activity is virtually absent in almost all normal somatic cells. As a result, cell senesce, leading to an eventual loss of graft function. Aging allografts, either due to cell injury related to transplantation and/or the use of organs from older donors, pose a threat to the long-term survival of a graft as constitutive cells of an aging organ have a much reduced ability to thrive after transplantation. In our study, human endothelial cells were found to undergo replicative senescence in culture with an increase in the percentage of senescent cells (beta-gal staining at pH 6) and a decrease in both the fraction of S-phase cycling cells and the proliferative index measured using CFDA-SE dye. Aging endothelial cells also demonstrated slow rates of proliferation and migration compared to younger cells. Unlike control cells that were transfected with an irrelevant gene vector, telomerase-transfected endothelial cells recovered rapidly after media replacement in cultures that had been serum starved for 2 weeks. Telomerase-transfected cells also retained a high proliferative index comparable to young cells as opposed to untransfected control cells. This young phenotype provided by telomerase expression through restoration of the telomeres may help to increase the longevity of organ transplants.

The effect of photodynamic therapy on rat urinary bladder with orthotopic urothelial carcinoma.

OBJECTIVE: To assess the effect of whole-bladder photodynamic therapy (PDT) on a rat model with orthotopic superficial bladder cancer, as PDT is an alternative intravesical therapy for treating superficial bladder cancer, based on an interaction between a photosensitizer and light energy to induce oxygen radicals that destroy tissue by lipid peroxidation. MATERIALS AND METHODS: In all, 76 female Fischer F344 rats were inoculated intravesically with AY-27 tumour cells. After establishing superficial tumour, 24 rats were treated with PDT using aminolaevulinic acid (ALA)-induced protoporphyrin IX as a photosensitizer, and a continuous-wave argon pumped-dye laser (638 nm). At 4 h after intravenous (300 mg/kg) or intravesical (100 mg/mL) administration of ALA the bladders were intravesically exposed to a 40 J/cm(2) light dose; 12 rats received no ALA but were exposed to the same light dose. Before administering ALA, urine cytology samples were taken for analysis. At 3 or 21 days the treated rats were killed and morphological changes in the bladder walls analysed by light microscopy. Forty rats served as controls to examine the presence of tumour. RESULTS: The tumour established in 33 of 40 rats (83%) in the controls, but after PDT with intravesical ALA there was carcinoma in only in one of 12 (P < 0.001, Pearson's chi(2) test). After PDT with intravenous ALA there was carcinoma in five of 11 rats (P = 0.063, Pearson's chi2 test). In the control group of 12 rats receiving only light energy there was carcinoma in three (P = 0.001, Pearson's chi(2) test). Histologically, at 3 days after PDT there was only mild superficial damage in all six rats treated intravesically. Bladder wall destruction reached the muscular layer, with an abscess in one of six rats treated intravenously. After 3 weeks of PDT there was muscular necrosis with perforation and abscess from catheterization two of six rats treated intravesically and in three the bladder wall totally recovered. In the intravenous group the bladder walls were normal or had only mild superficial damage. Cytology of the urine sediment failed to detect half the tumours in the treatment groups. CONCLUSION: These results support the use of PDT with intravesical ALA-induced protoporphyrin X for treating superficial bladder carcinoma. Intravesical was better than intravenous ALA in eradicating bladder carcinoma with PDT.

Gene therapy: a lipofection approach for gene transfer into primary endothelial cells.

Despite the great potential of gene therapy to become a new treatment modality in future medicine, there are still many limitations to overcome before this gene approach can pass to the stage of human trial. The foremost obstacle is the development of a safe, efficient, and efficacious vector system for in vivo gene application. This study evaluated the efficacy of lipofection as a gene delivery vehicle into primary endothelial cells. Transfection efficiency of several lipid-based reagents (Effectene, Fugene 6, DOTAP) was examined at experimental temperatures of 37 degrees C, 24 degrees C, and 6 degrees C. Human umbilical vein endothelial cells (HUVECs) were transfected with the enhanced green fluorescent protein (EGFP) using precise amounts of DNA (Effectene, 0.2 microg; Fugene 6, 0.5 microg; DOTAP, 2.5 microg) and lipids (Effectene, 10 microl; Fugene 6, 6 microl; DOTAP, 15 microl) optimized in our laboratory. Duration of incubation in the DNA/lipid transfection mixture varied for each lipid transfectant as follows: 5 h for both Fugene 6 and DOTAP and 3 h for Effectene. Efficiency of transfection was quantified by microscopic evaluation of EFGP expression in a minimum of 100 cells per group. Transfection efficiencies achieved with these lipofection agents were 34 +/- 1.3% (mean +/- SEM), 33 +/- 1.4%, and 18 +/- 1.5% for Effectene, Fugene 6, and DOTAP, respectively, at 37 degrees C. Transfection results were lower at 24 degrees C with mean efficiencies of 26 +/- 2.4% for Effectene, 14 +/- 2.9% for Fugene 6, and 15 +/- 3.2% for DOTAP. Furthermore, mean efficiencies at 6 degrees C were 6 +/- 0.5%, 8 +/- 1.5%, and 6 +/- 0.0% for Effectene, Fugene 6, and DOTAP, respectively. Efficiency of transfection appeared to be temperature dependent (ANOVA; p < 0.0001). In spite of a significant decrease (37 degrees C vs. 24 degrees C: p < 0.0001; 37 degrees C vs. 6 degrees C: p < 0.0001; 24 degrees C vs. 6 degrees C: p < 0.0115) in transfection efficiency at low temperatures, the successful in vitro gene manipulation renders lipofection a potential gene delivery strategy for in vivo gene therapy.

Selective cytotoxicity of gemcitabine in bladder cancer cell lines.

We have examined the cytotoxic effect of gemcitabine in intravesical therapy using an in vitro co-cultured spheroid model composed of transitional cell carcinoma (TCC) and fibroblasts from both human and rat species. Immunohistochemistry analysis of the co-cultured spheroids, using cytokeratin-13 and vimentin antibodies against TCC and fibroblasts, respectively, showed the central location of fibroblasts within the spheroid, whereas TCC formed the peripheral layers. Spheroids composed of human TCC and fibroblasts (MGH-U3/CRL-1120 or RT-112/CRL-1120) as well as rat TCC and their corresponding fibroblasts (AY-27/RF-Ed1) displayed the same drug tolerance profile after an exposure of 0, 1, 3, 5, 7 and 14 days. As confirmed by time-lapse photography, MTT essay and vital dye staining, gemcitabine selectively killed the human and rat bladder cancer cell lines, but did not affect un-transformed human and rat fibroblast lines.

Resection of renal cell carcinomas with inferior vena caval extension using deep hypothermic circulatory arrest.

Renal cell carcinoma with tumour thrombus extension into the inferior vena cava presents a difficult surgical challenge. The conventional surgical approach, which involves isolating the inferior vena cava, incising its wall and removing the thrombus, can have high incidences of perioperative mortality and embolization of the tumour thrombus compounded by severe hemorrhage. Four patients with renal cell carcinomas extending into the inferior vena cava were supported with cardiopulmonary bypass and deep hypothermic circulatory arrest during tumour excision. All of the operations were successfully performed with no mortality and minimal morbidity. The technique allowed the surgeon to operate in a bloodless field, thereby improving visibility and allowing complete tumour excision without significantly prolonging operative time. It is believed that this technique has improved the safety and technical feasibility of what had previously been a complicated and risky surgical procedure.

Light dosimetry using the P3 approximation.

In earlier work, we demonstrated that radiance, calculated using the P3 approximation in a plane wave geometry, could be used to accurately predict the optical parameters of an Intralipid/methylene blue phantom. Plane wave geometry is impractical for clinical use but the results of this work encouraged us to further develop the P3 approximation for a spherical geometry, described in this paper. Radiance predicted by this model for a defined Intralipid/methylene blue phantom was compared with radiance measured in this phantom. The results demonstrate that the spherical derivation of the P3 approximation will reproducibly predict optical parameters of a tissue phantom as effectively as the slab geometry derivation of the P3 approximation. In a similar protocol, the P3 approximation was used to estimate the optical parameters of ex vivo human prostate. Radiance in this case was measured in the prostate samples using an after loading technique. Three prostate samples tested were found to be surprisingly optically homogeneous. The after loading protocol described in this paper could form the basis of a minimally invasive and effective clinical method to optically characterize human prostate.

Breakdown of the isobaric multiplet mass equation at A = 33, T = 3/2.

Mass measurements on (33,34,42,43)Ar were performed using the Penning trap mass spectrometer ISOLTRAP and a newly constructed linear Paul trap. This arrangement allowed us, for the first time, to extend Penning trap mass measurements to nuclides with half-lives below one second ( 33Ar: T(1/2) = 174 ms). A mass accuracy of about 10(-7) (deltam approximately 4 keV) was achieved for all investigated nuclides. The isobaric multiplet mass equation was checked for the A = 33, T = 3/2 quartet and found to be inconsistent with the generally accepted quadratic form.

Acetochlor-induced rat nasal tumors: further studies on the mode of action and relevance to humans.

The herbicide acetochlor, and its analogue alachlor, have similar toxicological properties, the most significant being the induction of nasal adenomas in rats in 2-year feeding studies. Previous investigations have proposed a mode of action involving metabolism to a quinone-imine, the formation of protein adducts, cell death, and compensatory hyperplasia leading to the observed adenomas. Comparisons between rats and humans of the metabolic cascade leading to the quinone-imine indicate that these chemicals do not pose a threat to humans. Further investigations with acetochlor, presented here, have revealed an additional activation pathway in which a sulfoxide metabolite of acetochlor plays a key role. The sulfoxide was found to be the major plasma metabolite in rats dosed with acetochlor. Whole-body autoradiography studies established that this metabolite selectively accumulates and persists in the olfactory epithelium of rats. Radiolabeling of the sulfoxide molecule in the phenyl ring and in the sulfoxide side-chain demonstrated that the metabolite accumulating in nasal tissues retains the sulfoxide side-chain. The formation of a quinone-imine from the sulfoxide was facilitated by hydroxylation of the phenyl ring by a cytochrome P450 isoenzyme which was specific to the nasal epithelium in the rat. This metabolic conversion could not be detected in 33 fresh human nasal tissue samples, supporting the earlier view that the acetochlor-induced rat nasal tumors do not represent a hazard for humans.

Characterization of a novel transplantable orthotopic rat bladder transitional cell tumour model.

An animal tumour model that mimics the human counterpart is essential for preclinical evaluation of new treatment modalities. The objective of this study was to develop and characterize such a model. To accomplish this, the established AY-27 rat bladder transitional cell carcinoma (TCC) cell line was transplanted orthotopically into Fischer CDF344 female rats. AY-27 TCC cells were grown in monolayer cell culture and instilled intravesically as single cell suspensions into bladders that had been conditioned with mild acid washing. Tumour growth was assessed weekly by subjecting the rats to magnetic resonance imaging (MRI). At intervals following implantation and MRI tumour detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies. Flow cytometry was also performed for detection of Fas or Fas-ligand expression on AY-27 cells. The overall tumour establishment was 95% (97/102 rats) at 12-50 days, while in a subgroup of animals sacrificed at 16 days, 80 out of 82 animals (97%) developed TCC, the majority of which was superficial. Tumour stage was assessed by gross pathology and light microscopy. Histological examination of the tumour specimens confirmed the presence of grade II-III TCC. Immunocytochemistry confirmed that the tumour model maintained the features of TCC. The changes seen on MRI correlated well with the extent of tumour invasion identified histologically. Patchy carcinoma in situ could be detected histologically 12-13 days post-inoculation, and progressed to papillary tumour or invasive disease thereafter. Neither Fas nor Fas-ligand was expressed on AY-27 cells. The orthotopic AY-27 TCC model is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.

A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens.

Coprocessing of kininogens by a mixture of human mast cell tryptase and neutrophil elastase was explored as a potential substitute for the kallikrein-dependent pathway for kinin generation during inflammation. Tryptase easily excised bradykinin from the synthetic heptadecapeptide, ISLMKRPPGFSPFRSSR, but was unable to produce significant amounts of kinin by proteolysis of kininogens. However, a mixture of tryptase and elastase released bradykinin from each protein with a yield comparable to that of human plasma kallikrein. Significantly, neither plasma nor tissue kallikrein was able to effectively process N-chlorosuccinimide-oxidized high molecular weight kininogen, an effect attributed to the oxidation of a methionine residue upstream from the N terminus of the kinin domain. In support of these results the model heptadecapetide, ISL(MO)KRPPGFSPFRSSR, was also resistant to hydrolysis by either kallikrein. In contrast, the release of bradykinin from oxidized peptide or protein substrates by the tryptase/elastase mixture was not altered. Because kininogen modification may occur at inflammatory sites, as a result of the oxidative burst of recruited neutrophils and macrophages, these results suggest an alternative pathway for kinin production and the necessity for the novel utilization of two specific proteinases known to be released from these cells during inflammatory episodes.

Radiance modelling using the P3 approximation.

Light dosimetry is an essential component of effective photodynamic therapy (PDT) of tumours. Present PDT light dosimetry techniques rely on fluence-based models and measurements. However, in a previous paper by Barajas et al, radiance-based light dosimetry was explored as an alternative approach. Although successful in demonstrating the use of Monte Carlo (MC) simulations of radiance in tissue optical characterization, the MC proved time consuming and impractical for clinical applications. It was proposed that an analytical solution to the transport equation for radiance would be desirable as this would facilitate and increase the speed of tissue characterization. It has been found that the P3 approximation is one such potential solution. Radiance and fluence expressions based on the P3 approximation were used to optically characterize an Intralipid-based tissue phantom of varying concentration of scatterer (Intralipid) and absorber (methylene blue) using a plane wave illuminated, semi-infinite medium geometry. The results obtained compare favourably with the Grosjean approximation of fluence (a modified diffusion theory) using the same optical parameters (mu(a), mu(s), g). The results illustrate that radiance-based light dosimetry is a viable alternative approach to tissue characterization and dosimetry. It is potentially useful for clinical applications because of the limited number of invasive measurements needed and the speed at which the tissue can be characterized.

The reproductive toxicity of molinate and metabolites to the male rat: effects on testosterone and sperm morphology.

Molinate causes an impairment in reproductive capability in the male rat. Administration of molinate to rats (40 mg/kg/day for 7 days) caused a distinctive sperm lesion. At higher doses of molinate (140 mg/kg for 7 days) this lesion was accompanied by morphological changes to the testis that were consistent with a delayed release of the late spermatids to the seminiferous tubular lumen, a process controlled by the release of testosterone. In accordance with this, molinate (>/=40 mg/kg) caused a marked decrease in the concentration of circulating and testicular testosterone. The Leydig cells of the testis appear to be the primary target site in that radiolabel from [3H]molinate specifically localized within this cell type. In addition, esterase activity in the Leydig cells was inhibited following molinate administration. In vitro, molinate is a poor inhibitor of esterase activity, whereas molinate sulfoxide, a major metabolite of molinate in rats, and molinate sulfone were shown to be potent inhibitors of this process, suggesting that metabolic activation of molinate is required in vivo. Molinate sulfoxide (>/=10 mg/kg) caused an identical sperm lesion to that of molinate and markedly decreased plasma and testicular testosterone concentration. These effects were not seen with the molinate metabolites 4-hydroxymolinate (10 mg/kg), molinate sulfone (10 mg/kg), and hexamethyleneimine (10 mg/kg). Since the sperm lesion is a secondary event caused by a disruption of spermatogenesis, this would imply that the testis lesion and the reproductive impairment are also a consequence of molinate sulfur oxidation.

Elevated plasma endothelin-1 levels in sickle cell anemia: relationships to oxygen saturation and left ventricular hypertrophy.

Endothelin-1 (Et-1) is a vasoconstrictor produced by endothelial and vascular smooth muscle cells in response to hypoxia, which induces hypertrophy in cultured cardiac myocytes. We measured plasma Et-1 levels and left ventricular dimensions in 13 patients with sickle cell anemia (SCD) and in 12 African-American controls ages 16-29 years. Endothelin-1 concentrations are significantly higher in SCD subjects than controls (10.6 +/- 1.9 vs. 3.0 +/- 1.3 pmol/L). There was a negative correlation between oxygen saturation and Et-1 levels in SCD patients (r = -0.71, P = 0.01). SCD subjects have more dilated and hypertrophied hearts corrected for body surface area than controls as evidenced by significant increases in left ventricular end diastolic dimension (31 +/- 0.8 vs. 24 +/- 0.9 mm/m2, P < 0.001), left ventricular end systolic dimension (20 +/- 0.9 vs. 16 +/- 0.8 mm/m2, P = 0.002), left ventricular posterior wall thickness (5.0 +/- 0.1 vs. 4.0 +/- 0.1 mm/m2, P < 0.001), and left ventricular mass (125 +/- 7.2 vs. 69 +/- 5.1 g/m2, P < 0.001). The index of left ventricular function, the shortening fraction, was not different between groups (34 +/- 1.2% in SCD vs. 35 +/- 1.5% in controls). The correlation between left ventricular mass and levels of Et-1 in SCD subjects was not significant (r = 0.47, P = 0.121).

Biodistribution of Photofrin II and 5-aminolevulinic acid-induced protoporphyrin IX in normal rat bladder and bladder tumor models: implications for photodynamic therapy.

Photodynamic therapy (PDT) has been considered as a potential therapy for superficial bladder carcinomas. Cutaneous photosensitivity and reduction of bladder capacity are the two well-known complications following systemic administration of the commonly used photosensitizer, Photofrin II (PII). The objective of the present study was to evaluate whether intravesical (i.b.) instillation of photosensitizers for PDT of bladder cancer might be a more suitable treatment method. Female Fischer rats were utilized to develop orthotopic and heterotopic bladder tumor models. Rats bearing orthotopic bladder tumors were treated either intravesically or intravenously with graded doses of 5-aminolevulinic acid (ALA) or PII. Normal rats received the same doses of ALA or PII. As well, rats bearing heterotopic tumor were studied for comparison. The biodistribution times (times allowed for tissue uptake and bioconversion following drug administration) were 2, 4 or 6 h. Porphyrin fluorescence intensities within tumor, urothelium, submucosa, bladder muscularis and abdominal muscle were quantitated by confocal laser scanning microscopy. Following intravenous (i.v.) injection of ALA, tumor protoporphyrin IX (PpIX) levels peaked at 4 h and diminished by 6 h. The PpIX ratios of tumor-to-bladder mucosa, submucosa and muscle layers were 3:1, 5:1 and 8:1, respectively, 4 h following 1000 mg/kg ALA injection. After ALA instillation, the optimal biodistribution time appeared to be 4 h. Bladder instillation provided comparable tumor labeling with the i.v. route, but lost selectivity of PpIX accumulation between tumor and normal urothelium. The PpIX ratio of tumor-to-bladder muscularis was 5:1. After i.b. instillation of PII, porphyrin fluorescence was detected only within tumor and urothelium, while porphyrin fluorescence was mainly located in bladder submucosa following i.v. injection. Intravesical administration of ALA or PII might be feasible for PDT of superficial bladder cancers.

Monte Carlo modelling of angular radiance in tissue phantoms and human prostate: PDT light dosimetry.

Photodynamic therapy (PDT) is a promising technique for destroying tumours. Photosensitizing drugs presently available are not sufficiently tumour specific; hence, light dosimetry is required in order to control light exposure and thereby restrict cell kill to the target tissue to avoid damage to healthy tissue. Current light dosimetry methods rely on tissue optical characterization by fluence measurements at several points. Fluence-based tissue characterization is impractical for tumours in organs such as prostate where access by optical probes is limited and the tumours are highly optically inhomogeneous. This paper explores the potential of radiance-based light dosimetry as an alternative. Correlation is found between Monte Carlo simulation of radiance in a tissue phantom and radiance measurements made using a new radiance probe. Radiance is sensitive to variations in the tissue optical parameters, absorption coefficient mu(a), scattering coefficient mu(s), and anisotropy factor g, and therefore is potentially useful for tissue characterization. Radiance measurements have several advantages over fluence measurements. Radiance measurements provide more information from a single location, better spatial resolution of the tissue optical parameters, and higher sensitivity in discriminating between different media. However, the Monte Carlo method is too slow to be of practical value for tissue characterization by correlation of measured and simulated radiance. An analytical solution to the transport equation for radiance would be desirable as this would facilitate and increase the speed of tissue characterization.

Calpromotin, a cytoplasmic protein, is associated with the formation of dense cells in sickle cell anemia.

We have tested the hypothesis that dense cell formation in sickle cell disease is associated with increased binding of calpromotin to the membrane, an event that occurs during the activation of calcium-dependent potassium transport. By SDS polyacrylamide gel electrophoresis, we found that sickle cell membranes contained more calpromotin than did normal membranes when stained with Coomassie brilliant blue or when transferred to nitrocellulose paper and immunostained with horseradish peroxidase. Also, the membranes from dense sickle cells contained significantly (P = 0.00055) higher levels of calpromotin, 2.62+/-1.59 microg/mg membrane protein, compared to light sickle cells, 1.40+/-0.70 microg/mg membrane protein, when measured by an enzyme-linked immunosorbent assay. The ratio of calpromotin associated with dense cell membranes to light cell membranes was significantly greater than 1.0 (P < 0.00005). Transmission electron micrographs of immunogold-labelled membranes supported the increase in calpromotin binding in dense sickle cell membranes. In addition, the immunogold probe demonstrated clustering, which was not observed in light sickle cell membranes nor in normal membranes. Finally, we incubated HbSS cells in vitro using a repetitive deoxygenation/ reoxygenation procedure to produce dense cells and then measured the levels of calpromotin associated with their membranes. As expected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the incubation. The correlation coefficient, calculated between the increase in dense cell formation and the increase in calpromotin associated with the membrane, was statistically significant (P = 0.038). The results demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activation of the calcium-dependent potassium channel.

Preclinical assessment of hypocrellin B and hypocrellin B derivatives as sensitizers for photodynamic therapy of cancer: progress update.

Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomeric form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 [2-(N,N-dimethylamino)-propylamine-HB]. Extinction coefficients at 630 nm (epsilon 630) are 6230, 6190 and 4800, respectively; and 1O2 quantum yields, phi, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA-R1 permanently ablates EMT6/Ed tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhimurium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.

Protein 7.2b of human erythrocyte membranes binds to calpromotin.

Calpromotin is a soluble cytoplasmic protein of human red blood cells which is involved in the activation of the charybdotoxin-sensitive calcium-dependent potassium channel. This activation is associated with increased binding of calpromotin to the red cell membrane. To elucidate this mechanism we tested different fractions of red cell membrane proteins to bind to a calpromotin affinity column. Proteins, which bound specifically to the column, were eluted and identified by SDS polyacrylamide gel electrophoresis. This procedure demonstrated that spectrin and actin, from a low salt extraction of the membrane, bound weakly to the column and a portion of this could be attributed to non-specific binding. Glyceraldehyde-3-phosphate dehydrogenase (band 6) and a 40K molecular weight band, from a high salt extraction of the membrane, bound strongly to the affinity column. Several minor integral membrane proteins, obtained by Triton X-100 treatment of the membrane, bound specifically to the calpromotin affinity column. The molecular weight of these proteins ranged from 95k to 23K. We further demonstrated that the 31.5K band from this fraction is protein 7.2b (stomatin) by staining with a monoclonal antibody. Protein 7.2b is believed to have a role in regulating monovalent cation transport through the erythrocyte membrane.