4-Thiouridine incorporation into the RNA of monkey kidney cells (CV-1) triggers near-UV light long-term inhibition of DNA, RNA and protein synthesis.

Monkey kidney cells (CV-1) grown for 4 h in the presence of 0.1 mM 4-thiouridine (s4Urd) incorporate this photoactivable uridine analog in their RNA. A minor, 5-8%, thiolated RNA fraction can be isolated from bulk RNA by affinity chromatography. This RNA fraction contains 1.5-2.5 s4Urd residues per 100 nucleotides and exhibits a broad chain length distribution ranging from 700 to 7000 nucleotides. It is essentially of nuclear origin and amounts to 30% of the RNA synthesized during exposure of cells to s4Urd. Under the same s4Urd labeling conditions, no thiolated pyrimidine residues have been detected in DNA. Irradiation with 365 nm light (45 kJ/m2) of the cells immediately after s4Urd exposure triggers long-term inhibition of DNA, RNA and protein synthesis accompanied by a linear decline (50% in 2 days) in the total cell mass of cultured cells. In contrast, exposure to s4Urd alone results in moderate but reversible inhibitory effects. The available data suggest that s4Urd-induced photolesions in newly synthesized RNA such as RNA-RNA cross-links as well as RNA-protein bridges are directly involved in impairment of essential cellular functions.

Photosensitization of mitochondria by liposome-bound porphyrins.

We have compared the photodynamic activities of hematoporphyrin (HP) and protoporphyrin (PP) on isolated rat liver mitochondria by measuring the decline of the respiratory control ratio (RCR) after irradiation at 365 nm. Before addition to the respiratory medium, the dyes were dissolved in phosphate-buffered saline (PBS) or incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC), sometimes enriched with cholesterol (Chol) or cardiolipin (Card), which are naturally present in mitochondrial membranes. Chol and especially Card strongly increase the porphyrin uptake by mitochondria. In all experimental conditions, PP is taken up by mitochondria to a higher extent than HP. Nevertheless, under conditions giving the same amount of mitochondria-bound dye, HP is a more efficient photosensitizer than PP. As the efficiency of singlet oxygen production has been shown to be equivalent for the two porphyrins in monomeric state, the resulting photobiological effects are explained in terms of different localization of HP and PP in the mitochondrial membranes. In particular, HP preferentially localizes in the protein-rich polar domains of the inner mitochondrial membrane, whereas PP dissolves in the lipid regions of the membranes.

Factors influencing the distribution pattern of porphyrins in cell membranes.

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic study of the effects of psoralen and ultraviolet light on DNA, RNA and protein synthesis in cultured keratinocytes and psoriatic skin.

DNA, RNA and protein synthesis were studied by autoradiography in cultured keratinocytes, immediately, 24 and 48 h after 8-methoxypsoralen and ultraviolet light, (PUVA) treatment. Using the same technique, the immediate and long-term effects of PUVA therapy on DNA, RNA and protein synthesis were analysed in skin biopsies from psoriatic patients. In the cultures, immediately after irradiation, DNA and RNA syntheses were similarly inhibited in a dose-dependent manner, while protein synthesis was slightly affected only for the highest dose. After 24 h, RNA synthesis recovered whereas DNA synthesis was more severely inhibited suggesting that other cell components may be damaged by PUVA. In patients, DNA and RNA syntheses decreased immediately after PUVA sessions. During all the sessions until the psoriatic plaques had cleared, an impairment of DNA synthesis was observed in comparison with the synthesis in involved and uninvolved skin before treatment. These results suggest that the therapeutic efficiency of PUVA is based on the inhibition of DNA replication due to direct effects on nucleic acids but also to photoreactions with other cell components.

Photosensitivity of DNA replication and respiration to haematoporphyrin derivative (photofrin II) in mammalian CV-1 cells.

DNA synthesis, as well as respiration, has been studied in CV-1 cells incubated with 5 or 25 micrograms/cm3 haematoporphyrin derivative Photofrin II (PF II) for 1, 24 or 48 h and then irradiated with various doses of UVA light (365 nm). The impairments of DNA synthesis increased with the duration of incubation with the porphyrin, its concentration and the dose of irradiation. The cellular consumption of oxygen is also inhibited by the treatment, but less severely. In the case of the higher PF II concentration (25 micrograms/cm3), the impairment of DNA synthesis after illumination seems to be mainly due to 3HTdR transport inhibition. This effect can be related to plasma membrane damage as shown by lactate dehydrogenase leakage. At 5 micrograms/cm3 PF II, DNA synthesis inhibition is observed even after short exposure to PF II and light without 3HTdR transport impairment. In that case, DNA and/or mitochondrial photodamage may explain the inhibition.

4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells.

Monkey kidney cells (CV-1) cultivated in the presence of 0.1 mM 4-thiouridine (S4U) and subsequently illuminated at 365 nm exhibit a marked RNA synthesis inhibition. Maximal effect (approximately 40%) was obtained for a 4 h S4U incubation and a 45 KJ/m2 dose. Under these conditions up to 20% of total cellular RNA is retained at the interphase during phenol-chloroform extraction. The fraction of RNA crosslinked to proteins amounts to 50% of the 3H-uridine labeled RNA synthesized during S4U incorporation and less than 10% for the control samples. This strongly suggests that S4U incorporated within the RNA chains acts as a photoaffinity probe. The data above provide the basis of a method for studying in vivo RNA-protein interactions under non destructive conditions.

Effects of photodynamic action on energy coupling of Ca2+ uptake in liver mitochondria.

In mitochondria isolated from rat liver, incubated in the presence of 6 X 10(-3) mM hematoporphyrin and irradiated with UV light at 365 nm, respiration, oxidative phosphorylation and Ca2+ uptake were measured in order to determine the respective photosensitivity of these functions. Irradiation with increasing doses produces uncoupling of oxidative phosphorylation followed by inhibition of Ca2+ uptake and finally arrest of respiration. Ca2+ uptake stimulated by the addition of ATP was also studied in mitochondria uncoupled by irradiation which were still able to concentrate Ca2+ aerobically. Anaerobic Ca2+ uptake driven by ATP hydrolysis was found to be similar in control and in irradiated mitochondria, suggesting a different photosensitivity for the ATPase as compared to the ATP-synthase activity.

UV-induced unscheduled DNA synthesis in guinea pig skin melanocytes isolated in culture.

Pigmented melanocytes isolated in culture from the epidermis of guinea pig ears were used to study the unscheduled DNA synthesis (UDS) induced by ultraviolet (UV) irradiation at 254 nm. After irradiation, the cells were labeled for 6 hr with 3H-thymidine (3HTdR), fixed, dehydrated, and flat embedded in Epon. Radioautographs were made on serial 0.5-1 micrometer thick sections of the cultures in order to distinguish silver grains from pigment granules. Cells irradiated with 5, 10, or 50 joules (J) m-2 showed a 3HTdR uptake due to UDS, whereas their proliferative ability, measured by the 3HTdR uptake due to scheduled DNA synthesis, was very low. For the same UV doses, UDS was lower in melanocytes than in guinea pig fibroblasts and keratinocytes.

Studies on guinea pig skin cell cultures. V. Co-culture of pigmented melanocytes and albino keratinocytes, a model for the study of pigment transfer.

Mixed cultures of melanocytes (M) and keratinocytes (K) are easily obtained from pigmented guinea pig ear skin. They are suitable for the study of pigment transfer from M to K. However, quantitation is difficult because many K are already loaded with pigment prior to cultivation. A technique is presented in which pigment-producing M are co-cultured with K of albino origin. Pigmented guinea pig ear skin is split with trypsin and basal cells including M are harvested. The cell suspension is treated with sodium citrate which prevents the attachment of K (but not of M) to the culture substrate. Ninety per cent pure M cultures are obtained. Five to seven days later, another basal cell suspension is prepared, this time from albino ear skin. This second suspension is seeded on top of the pigment-forming culture of M. The number of contacts between albino K and pigment-forming M increases as a direct function of time. Contrarily, the number of K which become pigmented increases until the fifth day of co-culture and decreases thereafter.