Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5-hydroxyl termini and dephosphorylate 3-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.Transient DNA strand breaks and short gaps are frequently observed in cellular DNA. Many arise during regular cellular activity such as DNA replication, recombination, or differentiation. Others occur as a consequence of exposure to endogenous or exogenous DNA damaging agents. Repair of these strand interruptions is usually mediated by DNA ligases and polymerases. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by nucleases, such as DNase II, and many produced by ionizing radiation bear 3Ј-phosphate and 5Ј-hydroxyl groups (1-4), and therefore must be processed before they can be acted upon by DNA ligases or polymerases.One enzyme that possesses the capacity to both phosphorylate 5Ј-hydroxyl termini and dephosphorylate 3Ј-phosphate termini is polynucleotide kinase (PNK).1 The PNK from T4 phage has found widespread application in molecular biology, especially for radiolabeling DNA and oligonucleotides (5). It can act on DNA and RNA and even phosphorylate nucleoside 3Ј-monophosphates. However, the main cellular function of the T4 enzyme is not to repair DNA, but rather to counter the action of a phage endoribonuclease that cleaves tRNA (6). Eukaryotic PNKs fall into two categories depending on whether their preferred substrate is DNA or RNA (7). While both can phosphorylate 5Ј-termini, only the former have an associated 3Ј-phosphatase activity (8 -12).Mammalian DNA kinases have ...

Summary 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.

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.

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.

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.