Lack of Enantioselectivity of Herpes Virus Thymidine Kinase Allows Safer Imaging of Gene Delivery

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Gene Therapy (2003) 10, 2052–2058 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery

L Magrassi1, G Finocchiaro2, G Milanesi3, F Benvenuto4, S Spadari3 and F Focher3 1Neurosurgery, Department of Surgery, University of Pavia, IRCCS Policlinico S. Matteo, Pavia, Italy; 2Istituto Nazionale Neurologico Besta, via Celoria, Milano, Italy; 3Istituto di Genetica Molecolare, CNR, via Abbiategrasso, Pavia, Italy; and 4Laboratorio di Biotecnologie, IRCCS Policlinico S.Matteo, Pavia, Italy

Herpes simplex virus thymidine kinase (HSV-TK) is widely HSV-TK-positive cells inside a transplantable murine brain used in gene therapy. The enzymatic activity of HSV-TK may tumour after inoculation of cells producing retroviruses be traced in vivo by speciﬁc radiopharmaceuticals in order to carrying HSV-TK. Owing to their unnatural enantiomeric image transgene expression. However, most of these radio- conformation, phosphorylated LT metabolites are very pharmaceuticals are toxic per se or after activation by HSV- poorly processed by mammalian enzymes, thus leading to TK, and therefore do not represent ideal molecules for increased cellular retention and minimal toxicity. The ability clinical applications and repeated imaging. Unlike human to image cells expressing the HSV-TK gene by using radio- cytosolic TK, HSV-TK is not enantioselective and can labelled LT, without damaging the cells accumulating the efﬁciently phosphorylate both D and L enantiomers of phosphorylated L-nucleoside, will be important to monitor the b-thymidine. Here we show that, after phosphorylation by levels and spatial distribution of therapeutic vectors carrying HSV-TK, tritiated L-b-thymidine (LT) is selectively retained HSV-TK. inside the cells in vitro and in vivo. We used the in vivo Gene Therapy (2003) 10, 2052–2058. doi:10.1038/ accumulation of radioactive phosphorylated LT to image the sj.gt.3302112

Keywords: L-nucleosides; thymidine kinase; herpes virus; enantioselectivity

Introduction (Figure 1). LT is considered very safe, and neither in vitro nor in vivo studies have shown any sign of cellular or Noninvasive imaging of gene expression in vivo by mitochondrial toxicity.14,15 Here we demonstrate that LT positron emission tomography (PET) takes advantage is selectively accumulated in cells expressing HSV-TK of the enzymatic activity of reporter genes like herpes in vitro and in vivo without any apparent toxicity. We simplex virus thymidine kinase1,2 (HSV-TK) or the used tritium-labelled LT to identify HSV-TK-expressing sodium/iodide symporter.3 HSV-TK has been used as a cells autoradiographically in a transplantable brain therapeutic suicide gene in gene therapy trials.4–9 In vivo glioma tumour model. Furthermore, we also exploited imaging of cells infected by wild-type or modified herpes the transfer of phosphorylated LT from HSV-TK-expres- viruses, or cells transduced with HSV-TK containing sing cells to adjacent cells that do not express the vectors, has been performed in the past using nucleoside enzyme, to study the bystander effect.16 analogues that are preferentially metabolized by HSV-TK LT has the characteristics of an ideal substrate for and less efficiently by the corresponding mammalian tracing in vivo cells containing HSV-TK after infection enzyme(s).1,2,10,11 However, after phosphorylation, most or transfection, without any apparent toxicity-related of these molecules become a substrate for mammalian limitation on the number of repetitions of the analysis. nuclear and mitochondrial DNA polymerases, thus leading to mutagenesis and cell toxicity.12,13 Their toxicity makes them unsuitable or not recommended for Results repeated in vivo imaging of HSV-TK-producing cells, thus limiting the usefulness of HSV-TK as a reporter LT is efficiently metabolized by HSV-TK in vitro and gene. in vivo Based on the lack of enantioselectivity of HSV-TK in In order to test whether only the HSV-TK expressed in 14 comparison with mammalian TK, we used L-thymidine mammalian cells (and not endogenous human and (LT), the enantiomer of the naturally occurring murine TKs) efficiently phosphorylates 3H-L-b-thymi- D-thymidine, in order to trace HSV-TK-containing cells dine (3H-LT), we assayed TK activity in whole-cell without interfering with cell metabolism and survival extracts of human and murine cell lines using both D- and L-3H-thymidine as substrates. All cells were tested under optimal growth conditions in which mammalian Correspondence: Dr L Magrassi, Neurosurgery, Department of Surgery, University of Pavia, IRCCS Policlinico S. Matteo P.zle Golgi 2, 27100 TK is highly expressed. UTK cell extracts containing 3 Pavia, Italy active HSV-TK phosphorylated both H-D-b-thymidine Received 14 December 2002; accepted 30 May 2003 (3H-DT) and 3H-LT (Figure 2a), whereas the soluble Imaging HSV-TK with L-thymidine L Magrassi et al 2053

Figure 1 Metabolism of D- and L-thymidine. L-b-Thymidine (LT), the enantiomer of the naturally occurring D-b-thymidine (DT), is characterized by the presence of the unnatural sugar 20-deoxy-L-ribose in place of 20-deoxy-D-ribose. LT is a poor- or nonsubstrate for mammalian TK, but it is efficiently phosphorylated by HSV-TK14 using ATP as a phosphate donor. Once the unnatural L-thymidine monophosphate is formed inside the cells, di- and triphosphates are generated by poorly characterized membrane-bound kinases.18,19 b-L-Nucleotides are very poor- or nonsub- strates for mammalian DNA polymerases24,25 and remain trapped inside Figure 2 Assay of TK activity in HSV-TK-expressing and -nonexpressing the cell because their increased charge inhibits their passive leakage. cells. (a) TK activity was assayed on extracts of 106 human glioblastoma Hu197 cells and an equivalent number of UTK cells (a stable clone derived by infecting Hu197 cells with a retrovirus expressing HSV-TK). The addition of 3H-DT to the extracts allowed us to measure the enzymatic extracts derived from Hu197 cells, the UTK parental cell activity of both mammalian and HSV TKs, whereas the addition of 3H-LT 3 only allowed the measurement of HSV-TK activity. The values of line, were unable to phosphorylate H-LT despite their 3 high level of mammalian TK activity (Figure 2a). mammalian TK activity in UTK cells are those obtained using H-DT as substrate minus those obtained using 3H-LT. The experiments were Negative results were also obtained by assaying extracts performed in triplicate and the error bars refer to the standard deviation from another human glioblastoma cell line (U251), one (s.d.) of the results. (b) Radioactivity linked to phosphorylated murine glioma cell line (GL261) and one human L-nucleotides in Hu197 human glioblastoma cells, HepG2 human hepatocarcinoma cell line (HepG2): none of these lines hepatoma cells and AmTK cells after 1 or 24 h exposure to 3H-LT (175 nM). The experiments were performed in triplicate and the error bars express HSV-TK, although they do express high levels of 3 the mammalian cytoplasmic TK (data not shown). refer to the s.d. HepG2 cells are more active in phosphorylating H-LT than brain tumour-derived Hu197 cells, but after 24 h, the levels of 3H-LT- We also tested whether intact human and murine cells derived nucleotides in the cells not expressing HSV-TK are at least one 3 could phosphorylate H-LT by TK-independent mechan- order of magnitude less than those in the cells expressing the viral enzyme. isms after prolonged exposure to the drug in vitro. Hu197 As previously shown in HeLa cells, most of the radioactivity is associated cells, HepG2 and AmTk cells were maintained in vitro with 3H-LT mono-, di- and triphosphates.15,27 in the presence of 3H-LT (175 nM) and, after 1 or 24 h, extensively washed in 3H-LT free medium, in PBS and finally collected. The levels of total and phosphorylated AmTK cells and human glioblastoma Hu197 cells with 3 m 3H-LT were determined by counting the radioactivity H-LT (1.75 M) for 30 min. After incubation, the medium retained on DE-81 cellulose filters before and after was aspirated and the cells were washed three times in PBS before the addition of new medium without 3H-LT. washing in ammonium formate, which removes all 5 uncharged nucleosides and leaves nucleotides on the Serial aliquots (10 cells) were collected immediately after washing, and 30 min and 1, 2, 12 and 24 h after filter. We found no difference in the radioactivity 3 retained on the filters before and after formate washing. exposure to H-LT. The cells in each sample were lysed This indicates that unphosphorylated 3H-LT is quickly and their total radioactivity content measured. As shown purged from the cells during washes in fresh medium in Figure 3, the HSV-TK-expressing cells contained significantly more radioactivity at all times. Even 24 h and PBS. As shown in Figure 2b, the level of LT-derived 3 nucleotides was always at least one order of magnitude after exposure to H-LT, the levels of radioactivity in higher in the cells expressing HSV-TK than in those with HSV-TK-containing cells were still one order of magni- no HSV-TK expression. However, in line with the results tude higher than in the cells not expressing the viral of Hernandez-Santiago et al,15 the HepG2 cells (which enzyme. do not express HSV-TK) can, albeit inefficiently, phosphorylate 3H-LT after prolonged exposure. Autoradiography distinguishes HSV-TK-positive from HSV-TK-negative cells Retention of phosphorylated 3H-LT inside Definitive proof that mammalian cells expressing HSV-TK-positive cells HSV-TK can be visualized after exposure to 3H-LT comes In order to study differences in the production and from the following experiment. We grew AmTK and retention of 3H-LT metabolites in cells containing HSV- Hu197 cells to semiconfluence on polylysine-covered TK and in those that do not, we separately incubated slides. The cells were exposed to 3H-LT (87.5 nM),

Gene Therapy Imaging HSV-TK with L-thymidine L Magrassi et al 2054

Figure 3 After phosphorylation, 3H-LT is retained in cells expressing HSV-TK. Hu197 human glioblastoma cells and Am-TK cells were exposed to 3H-LT (1.75 mM) for 30 min, and then thoroughly washed and maintained in fresh medium under normal culture conditions. After 0, 30, 60, 120 and 3600 min, equal cell samples (105) were collected and total radioactivity determined by scintillation counting. The experiments were performed in triplicate and the error bars refer to s.d. Only the Am-TK cells constitutively expressing HSV-TK retained signiﬁcant amounts of radioactivity after 30 min: 52% of the radioactivity in the cells at time 0 was still present after 24 h.

washed three times with fresh medium and then further incubated in complete medium without 3H-LT for 1 h; at the end of the incubation period, they were again thoroughly washed in PBS, air dried and processed for autoradiography. After various exposure times, the slides were developed and counterstained. In agreement with the results of the experiments on whole-cell extracts, only the HSV-TK-expressing cells showed signiﬁcant labelling (Figures 4a and b), with the silver granules being mainly concentrated over the cytoplasm. This and the previous experiment suggest that, although 3H-LT can enter the cells, its phosphorylated metabolites become membrane-impermeable and can be used as a marker of HSV-TK-expressing cells.

Multiple exposures to 3H-LT do not affect cell growth and survival Both parental Hu197 and derived UTK cells expressing Figure 4 Phosphorylation of 3H-LT by HSV-TK allows autoradiographic HSV-TK were grown to semiconfluence, exposed for 24 h identification of HSV-TK-positive cells. (a) Haematoxylin-counterstained 3 autoradiographic image of Am-TK cells growing in a slide chamber after to H-LT (350 nM), washed and divided in three fractions 15-min exposure to 3H-LT (87.5 nM), extensive washing and autoradio- 5 (approximately 10 cells in each fraction). One fraction of graphy. Note that the density of the autoradiographic granules is higher the cells was replated and allowed to grow until it again in the cytoplasm than in the nucleus, as expected from the cytoplasmic reached semiconfluence, one-third of the cells was location of HSV-TK. An average number of 118721 granules was collected and the levels of 3H-LT retention determined associated with each cell; this number exceeded the average number of by scintillation counting as previously described, the last granules associated with human cells not expressing HSV-TK by one order of magnitude (see (b)). Scale bar: 5 mm. (b) Haematoxylin-counterstained fraction was plated in 24-well plates and the growth autoradiographic image of Hu197 human glioblastoma cells grown in a index after 72, 96 and 120 h in culture was determined by slide chamber. Hu197 cells do not express HSV-TK and are unable to MTT analysis.16,17 After 1 week from the initial exposure phosphorylate 3H-LT. The cells were exposed to the same concentration of to 3H-LT, the exposed cells – which were replated – 3H-LT and processed as in the case of the Am-TK cells described in (a). An 7 reached semiconfluence and were exposed again to average number of 6 5 granules was associated with each cell, a value 3H-LT. After 24 h of exposure, they were washed and that is indistinguishable from the background (the same cells processed for autoradiography without previous 3H-LT exposure). Scale bar: 5 mm. divided as described above. The entire growth-exposure and analysis cycle was repeated three times. As a control, 3 identical experiments were performed on Hu197 and H-LT phosphorylation reveals the presence of UTK cells without exposure to 3H-LT. Growth curves of HSV-TK cells in vivo the Hu197 (Figure 5a and b) and UTK cells obtained after In order to verify whether 3H-LT could efficiently be used multiple exposure to 3H-LT did not differ from those of in vivo to identify HSV-TK-expressing cells against a controls not exposed to 3H-LT. At every exposure, UTK background of nonexpressing cells, we stereotactically cells expressing HSV-TK were able to phosphorylate implanted 5 Â 104 GL261 murine glioma cells alone or and retain 3H-LT at a level more than 400 times mixed with 1 Â 104 AmTK cells into the left striatum of (9.37 þ 1.72 pmol 3H-LT/106 cells) that retained by C57BL6J mice. After 1 week, the engrafted mice received the Hu197 cells not expressing HSV-TK (0.02 þ 0.02 pmol an intraperitoneal injection of 22 nmol of 3H-LT and, 3H-LT/106 cells). 12 h later, were transcardiacally perfused under deep

Gene Therapy Imaging HSV-TK with L-thymidine L Magrassi et al 2055

Figure 5 Growth of Hu197 and UTK after multiple exposures to 3H-LT. (a) Results of MTT analysis performed 72, 96 and 120 h after plating Figure 6 In vivo analysis of HSV-TK-expressing cells. (a) Autoradio- Hu197 cells (white bars) or Hu197 cells previously exposed three times to graphic image (DIC optic) of a 30 mm thick cryostat section without 3H-LT (350 nM) (black bars), the interval between each exposure was counterstaining; two labelled cells are indicated by the arrows. GL261 cells 6 days, the length of the exposure was 24 h. Absorbance readings were and Am-TK cells were cografted at a ratio of 5:1 into the left striatum of a performed at 550 nm; these values are proportional to the growth of the C57BL6J mouse. At 1 week after the inoculum and 12 h after an i.p. cells. The experiments were performed in triplicate and the error bars refer injection of 3H-LT (22 nmol), mice were perfused with cold ringer lactate to s.d. (b) Results of MTT analysis performed 72, 96 and 120 h after containing heparin and exsanguinated. The brain was collected, frozen in plating UTK cells (white bars) and UTK cells previously exposed three isopentane cooled in liquid nitrogen and prepared for autoradiography as times to 3H-LT (350 nM) (black bars), the interval between each exposure described in the Materials and methods section. Scattered positive cells are was 6 days, the length of the exposure was 24 h. Absorbance readings were visible (arrows); most of the granules are concentrated at the cell periphery performed at 550 nm; these values are proportional to the growth of the outside the cell nucleus. Scale bar: 5 mm. (b) GL261 mouse glioma cells and cells. The experiments were performed in triplicate and the error bars refer Am-TK cells were cografted at a ratio of 5:1 into the left striatum of to s.d. No significant alteration in the growth of Hu197 and UTK cells is C57BL6J mice. Weighted samples of brain, kidney, liver and striated visible after multiple exposure to 3H-LT (350 nM). muscle (quadriceps) were collected, and the total radioactivity present in each sample was determined. The values were normalized against the radioactivity present in the kidney in order to allow interanimal anaesthesia with heparinated cold PBS until completely comparisons by correcting for differences in 3H-LT absorption and exsanguinated. Their brains were quickly dissected and excretion. Radioactivity in kidneys of control animals was 9.27 pmol/g of frozen in isopentane cooled in liquid nitrogen, and tissue, while in animals receiving the inoculum of the Am-TK cells samples of other tissues (liver, kidney and skeletal muscle) radioactivity was 4.6 pmol/g of tissue. The height of each column were also collected and immediately frozen. One brain represents the average values of three different samples from two animals; from each group of mice was cut using a cryostat into the error bars show the s.d. The radioactivity in the brain of the animals m grafted with GL261 and Am-TK cells was significantly (Po0.05) higher 30- m-thick serial sections, which were collected on poly- than that found in the brain of the animals not receiving the transplant. lysinated slides. After the sections containing the growing tumour had been identified, the adjacent slides were processed for autoradiography. After variable exposures, expressing HSV-TK to neighbouring cells with no HSV- the slides were developed and studied by difference TK expression, since this would be useful for investigat- contrast microscopy without counterstaining. Only sec- ing the ‘bystander effect’. This effect, which has been tions coming from the animals receiving the implant of demonstrated in vitro and in vivo, is responsible for the both AmTK and GL261 cells showed autoradiographically death of cells that do not express HSV-TK but are in labelled cells (Figure 6a). This finding suggests that direct contact with cells expressing the viral enzyme HSV-TK cells can also be identified by their ability 3 when the cells are treated with ganciclovir or other to phosphorylate H-LT in vivo.Wealsomeasuredthe nucleoside analogues selectively activated by HSV-TK.18 radioactivity in the brains and other organs of the grafted The bystander effect is thought to be due to the passage animals. As expected, a small but significant increase in of ganciclovir phosphate through gap junctions after the radioactivity was found in the brains of the mice receiving HSV-TK phosphorylation of ganciclovir.19 UEG1a cells the combined graft of GL261 and AmTK cells (Figure 6b). stably expressing an enhanced version of the green fluorescent protein (eGFP) and UTK cells expressing Transfer of phosphorylated 3H-LT highlights HSV-TK were independently incubated for 30 min in the the ‘bystander effect’ presence of 3H-LT (1.75 mM). After extensive washing, the We also studied in mixed cultures the extent of the cells of the two lines were harvested, thoroughly mixed transfer of 3H-LT radioactive metabolites from cells and plated. After 24 h, the cells were harvested again,

Gene Therapy Imaging HSV-TK with L-thymidine L Magrassi et al 2056 expression in vivo because it is more sensitive, provides greater contrast and offers lower background levels.22 Positron emission tomography with 124I-labelled FIAU has successfully been used to image cancer cells infected with herpes simplex viruses in human patients.1,2 FIAU phosphorylation, as in the case of other nucleosides currently used for imaging cells with HSV-TK activity, leads to significant toxic effects on HSV-TK-expressing cells.12 The FIAU monophosphate generated by HSV-TK action is a good substrate for further phosphorylation leading to FIAU triphosphate, which enters nuclear and mitochondrial DNA during replication and repair, and Figure 7 The bystander effect (transfer of 3H-LT metabolites). UEG1a cells causes mutagenesis and cell death.12,13 Finally, the use express eGFP and can be unambiguously identified by their strong green of FIAU as an antiviral agent in human patients led fluorescence under appropriate blue light illumination. UTK cells are not 23 fluorescent but stably express HSV-TK and can efficiently metabolize to severe toxicity and it has been banned. FIAU toxicity 3H-LT. Separated and mixed cultures (1:1 ratio) of the two clones were in cells not expressing HSV-TK is mainly due to FIAU cultured until confluence, after which the cultures were exposed to 3H-LT phosphorylation by mitochondrial TK25 and the in- (1.75 mM) for 1 h, washed with three changes of fresh medium and corporation of FIAU triphosphate into mitochondrial maintained for 24 h under normal culture conditions before harvesting. DNA by DNA polymerase gamma.13,25 The cells were sorted on the basis of their fluorescence using a LT can be phosphorylated by mitochondrial TK,26 but FACSvantage flow cytometer (Becton-Dickinson, San Jose, CA, USA). Radioactivity was separately determined in fluorescent (UEG1a) and to a much lesser extent than its natural D enantiomer. nonfluorescent (UTK) cells. The histogram on the left (a) shows the data However, because of its L conformation, the triphosphate from the HSV-TK-positive nonfluorescent UTK cells, and (b) the data from derivative is a very poor substrate for nuclear and the HSV-TK-negative fluorescent cells. The first column of both histograms mitochondrial DNA polymerases,27,28 and does not cause represents the 3H-LT and metabolites present in both clones when cultured any sign of mitochondrial toxicity even after prolonged separately. As expected, after 24 h, only the UTK cells retained a treatment.15 Our data suggest that radioactively labelled significant amount of radioactivity indicating 3H-LT phosphorylation. The second column represents the radioactive contents of the same clones LT could be used in vivo to image cells expressing HSV- maintained in mixed cultures; the radioactive content of the UEG1a cells is TK or other nucleotide kinases with relaxed enantios- significantly greater, thus indicating the transfer of 3H-LT metabolites electivity, without damaging the cells in which the from the UTK cells (bystander effect). phosphorylated LT metabolites are accumulated. Clinical trials testing LT as a potential drug against hepatitis B and the two populations sorted by FACS on the basis virus are currently in progress and no signs of toxicity 15 of the presence or absence of eGFP fluorescence. have been reported. Appropriate radioisotopic label- 11 15 Measurements of the radioactive nucleotide levels in ling of LT (e.g. with Cor O) should allow its use as a the sorted populations showed that a significant amount tracer in PET studies, and its lack of toxicity should allow of phosphorylated 3H-LT passed from the UTK to the repeated independent imaging of the same cells. The UEG1a cells when the two populations were grown ability to image cells carrying the HSV-TK gene by using together after labelling (Figure 7). UEG1a cells kept in a radiolabelled LT without generating toxic metabolites mixed culture with prelabelled UTK cells for 24 h in the cells accumulating the phosphorylated substrates contained 13.5 times more radioactivity than the same will be important for monitoring the levels and the cells maintained in isolation. spatial distribution of therapeutic vectors containing the viral gene as a suicide gene. Furthermore, the possibility of repeated imaging provided by LT will be even more Discussion interesting for applications where HSV-TK will be used as a reporter and not as a suicide gene. HSV-based Our findings indicate that LT is selectively metabolized vectors have been used successfully in experimental by cells expressing HSV-TK and that its metabolites are animals to treat nonneoplastic conditions like neuro- retained for prolonged periods of time. Purified human degenerative diseases,29 intractable pain30 and arthritis.31 cytoplasmic TK is unable to phosphorylate LT to any The TK gene has been deleted from most of the HSV significant extent.14 These data together with our vectors used for non-neoplastic applications. TK dele- negative results with HepG2 cell extracts, tested under tion, however, was only a step in the strategy adopted optimal conditions for human cytoplasmic TK activity, for the construction of the vector, and reintroduction of suggest that background LT phosphorylation observed the TK gene is not inconceivable. For all applications in intact cells15 is due to mechanisms independent of requiring long-term persistence of the therapeutic con- cytoplasmic TK. Similarly, L-nucleoside diphosphates are struct and not cellular suicide, LT will be very valuable phosphorylated to triphosphates by pathways different for repeated imaging of the cells containing the active from those responsible for D-nucleoside di- vector without jeopardizing their survival. phosphate phosphorylation.20,21 The limited extent of the background phosphorylation of LT in cells not Materials and methods transduced by HSV-TK-containing vectors15 does not interfere with the histoautoradiographic imaging of Materials the cells expressing HSV-TK. Upon our request, the LT was 3H-labelled in the 5-methyl Recent studies have shown that 124I- or 131I-labelled group of the thymine by CEO SibTech, Inc. (Newington, 20-fluoro-20-deoxy-1-beta-D-arabinofuranosyl-5-iodo- CT, USA) at a specific activity of 12 Ci/mmol; 3H-DT was uracil (FIAU) is the best probe for imaging HSV-TK obtained by ICN at a specific activity of 25 Ci/mmol.

Gene Therapy Imaging HSV-TK with L-thymidine L Magrassi et al 2057 The human Hu197, U251, and murine GL261 high- repeated centrifugation (400 g for 10 min at 41C). Cells grade glioma cell lines, PA317 and HepG2, and the were then disrupted in 5 volumes of distilled water by clones obtained from these cell lines were all cultured freezing and thawing in order to extract the nucleosides in DMEM supplemented with 10% heat-inactivated FBS and nucleotides. Aliquots of the suspensions were (GIBCO/Invitrogen) and antibiotics (penicillin, strepto- vortexed and immediately spotted on 23 mm DE-81 mycin and amphotericin B) at 371C in a 5% CO2 filters, which were washed three times in an excess of atmosphere. An amphotropic retrovirus producer cell 1mM ammonium formate, pH 5.6, in order to remove line derived from murine Am12 cells transfected with unconverted nucleoside, and then once in ethanol. After pSTK32 was generated (manuscript in preparation). From drying, the filters were immersed in 1 ml of scintillation the obtained clones, we selected for further studies fluid (Betamax, ICN, UK) and their radioactivity was Am12pSTK#8 (AmTK), a clone expressing HSV-TK and determined by scintillation counting. efficiently producing retroviruses capable of transducing the HSV-TK cDNA into human and rodent cells. The 3 UTK cells expressing HSV-TK were derived by retroviral Assaying H-LT toxicity after repeated exposures infection of Hu197 cells; after infection, the cells were Hu 197 and UTK cells were grown, plated into multiwell m (6) plates and grown until they reached semiconfluence. selected and maintained in G418 (400 g/ml) supple- 3 mented medium. The UEG1a cells, obtained by poly- They were then exposed to H-LT (350 nM), and after 24 h they were extensively washed in PBS. After further ethylenimmine-mediated transfection of plasmid 3 pEGFP-N1 (Clontech, Palo Alto, CA, USA) in Hu197 incubation for 1 h in fresh medium without H-LT, cells m were trypsinized and collected. One-third of the cells cells, were selected and maintained in G418 (400 g/ml) 5 supplemented medium. The C57BL6J mice were main- (3 Â 10 ) were used for determination of radioactive tained and experimented upon according to the National nucleotide content as previously described, one-third Institutes of Health guidelines and the Italian law (DL was replated into six-well plates and one-third was 116/92) governing the care and use of experimental divided and plated into a 24-well plate. Cells in the animals. The GL261 cells (5 Â 104) alone or mixed with 24-well plate were maintained under optimal growth Am-TK (1 Â 104) cells were stereotactically implanted conditions (see above) and their growth was determined by an MTT assay 72, 96 and 120 h after plating. The MTT into the left striatum of C57BL6J mice as previously 16 17 described.33 assay was performed as described (MTT was used at a final concentration of 1 mg/ml). Absorbance readings were performed at 550 nm with 630 nm as a reference Assay for thymidine phosphorylating activity in cell wavelength. The entire growth-exposure and analysis extracts cycle described above was repeated three times. As a A total of 106 cells were collected from subconfluent control, identical experiments were performed on Hu197 cultures of the different cell lines by trypsinization, and UTK cells but omitting 3H-LT. washed with ice-cold PBS and centrifuged. The pellet was resuspended in 5 volumes of 20 mM Hepes-K þ ,pH 7.5, 1 mM DTT, 0.5 mM PMSF. After 30 min on ice, the Histology and autoradiography cells were disrupted by mini-dounce homogenization Cells growing on slides, after extensive washings in PBS, and centrifuged at 10 000 g for 10 min at 41C. TK activity were air dried, dipped into the autoradiography emul- was assayed by incubating the supernatants at 371C for sion (Hypercoat, Amersham-Parmacia Biotech) and þ 1 20 min in a mixture containing 30 mM HEPES K ,pH exposed for 30 days at 4 C in a dark box in order to detect the radioactive signal. 7.5, 6 mM MgCl2,6mM ATP, 0.5 mM dithiothreitol (DTT), and 2 mM 3H-DT (2200 cpm/pmol, ICN) or 2 mM 3H-LT The mice were transcardially perfused under deep (900 cpm/pmol) in a total volume of 25 ml. The reaction anaesthesia with cold Ringer lactate containing heparin. was terminated by spotting 20 ml of the incubation After complete exsanguination, the brain was dissected, mixture on a 23 mm DEAE paper disk (DE-81, Whatman, frozen in isopentane cooled with liquid nitrogen and cut Maidstone, UK). The disk was washed three times in an into 20 mm thick sections using a cryostat. The sections excess of 1 mM ammonium formate, pH 5.6, in order to were fixed and dried in saturated paraformaldehyde remove any unconverted nucleoside, and then once in vapours, dipped into the autoradiography emulsion and ethanol. After drying, the filters were immersed in 1 ml exposed for a minimum of 30 days at 41C in a dark box in of scintillation fluid (Betamax, ICN, UK) and radio- order to detect radioactivity. activity was determined by scintillation counting. The total protein concentration in the supernatants Determination of labelled LT metabolites in animal was determined using the BIO-RAD protein assay tissues (BIO-RAD, Mu¨ nchen, Germany). One TK unit is defined After perfusion and complete exsanguination of the as the amount of enzyme catalysing the formation of animal as described above, weighed samples of brain, 1 nmol of TMP in 1 h at 371C. liver, kidney and striated muscle were collected and immediately frozen in liquid nitrogen. After thawing, the Determination of radioactive nucleotide content in cell organ fragments were solubilized for 6 h at 551Cin extracts 0.5 ml of Lumasolve (Lumac System AG, Basel, CH, After the cultures were exposed to 3H-LT for the USA) in order to extract the nucleosides and nucleotides. specified time periods, the cells were washed three times The suspensions obtained were centrifuged for 10 min at with cold PBS, detached from the plate using trypsin- 10 000 g, and the supernatants were collected. The EDTA, collected in fresh medium supplemented with amount of radioactivity present in 100 ml of each super- serum and washed again three times in PBS by means of natant was counted in a b-counter in 1 ml of Ecolume

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