Helen H. Evans

Photodynamic therapy (PDT) is dependent on the uptake of a photosensitizing dye, often a porphyrin-related macrocycle, by the tumor or other abnormal tissue that is to be treated, the subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the dye, and molecular oxygen to generate reactive oxygen intermediates. The initial oxidative reactions lead to damage to organelles in which the dye is bound, culminating in cell death and destruction of the tumor or abnormal tissue. Apoptosis is a common mechanism of cell death after PDT both in vitro and in vivo. PDT also triggers the activation of several signal transduction pathways in the treated cells; some of these are stress responses aimed at cell protection, while others are likely to contribute to the cell death process. The photosensitizers of greatest interest in PDT bind to various cytoplasmic membranes but are not found in the nucleus and do not bind to DNA. Nevertheless, some DNA damage is produced that can lead to mutagenesis, the extent of which is dependent on the photosensitizer, the cellular repair properties and the target gene. Thus, in spite of generating some responses common to ionizing radiation and other oxidative stresses, PDT is unique in the subcellular localization of damage, the combination of signaling pathways that are activated, and rapid kinetics of the induction of cell death processes.

The mode of cell death of two strains of mouse lymphoma L5178Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmission electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

STUDY DESIGN: This study examined how the culture system and region of cellular origin affect disc cell morphology and extracellular matrix production. OBJECTIVE: To determine the role of the cell populations in the different regions of the adult intervertebral disc in maintaining gradients in composition across the disc. SUMMARY OF BACKGROUND DATA: It is not known whether the steep profiles in composition across the intervertebral disc are maintained by distinct cell populations or whether differences in cell metabolism are determined by changes in the physical environment across the disc. Very little information exists on the matrix produced by cells from the mature, non-notochordal nucleus pulposus. METHODS: Cells were extracted from articular cartilage, nucleus pulposus, and the inner and outer anulus fibrosus of caudal discs from 18- to 24-month-old steers cultured in alginate or collagen gels or in monolayer. The effect of culture system and cell origin on cell morphology and matrix synthesis was measured using 35S-sulphate labeling and indirect immunolocalization. RESULTS: Distinct morphologic differences between cells from different regions cultured in monolayer were retained through two passages. The rate of sulfate incorporation varied with cell type. Immediately after isolation, it was two- to threefold greater for nucleus cells than for cells from the disc inner anulus or articular cartilage. The rate was lowest for outer anulus cells. It also varied with culture system. For all cell types, the incorporation rate was highest in alginate and lowest in monolayer. Immunolocalization showed that nucleus cells stained strongly for all proteoglycan epitopes, whereas outer anulus cells stained least and in monolayer produced little proteoglycan. CONCLUSIONS: The disc has at least three distinct cell populations, which differ in morphology and in amount and type of matrix they produce. Cells from mature nucleus pulposus produced sulfated glycosaminoglycans at a high rate in contrast to reported results for notochordal nucleus cells. Alginate, although an appropriate culture system for inner anulus and nucleus cells, may not be a suitable medium for outer anulus cells.

Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities to the cytotoxic effects of various mutagenic agents--i.e., strain LY-S has been found to be more sensitive, less sensitive, or similarly sensitive to individual agents in comparison to strain LY-R. Nevertheless, strain LY-S has been found to be uniformly less mutable than strain LY-R at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus following treatment with x-radiation, UV radiation, or alkylating agents. In the present work we have isolated subclones of strains LY-R and LY-S that are heterozygous at the thymidine kinase (Tk) locus (chromosome 11). We have found that a heterozygous LY-S Tk+/Tk- strain shows as high or higher mutability at the Tk locus than do heterozygous LY-R strains following treatment with x-radiation, UV radiation, or ethyl methanesulfonate. Mutability of all heterozygous strains at the Tk locus is much higher than at the Hprt locus following treatment with these mutagenic agents, with the exception of one heterozygous LY-R strain that possesses only one chromosome 11 and that is poorly mutable at the Tk locus by x-radiation. On the basis of these results, we have suggested that because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; multilocus lesions lead to poor recovery of viable mutants when the target locus is closely linked to essential genes and situated on a hemizygous chromosomal region (e.g., the Hprt locus on the X chromosome or the Tk locus in strains monosomic for chromosome 11); and x-radiation is a relatively poor mutagen at loci situated on hemizygous chromosomal regions, in repair-efficient and repair-deficient cells, because of its tendency to form multilocus lesions.