The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD.

BACKGROUND: Goblet cell metaplasia, a common feature of chronic obstructive pulmonary disease (COPD), is associated with mucus hypersecretion which contributes to the morbidity and mortality among patients. Transcription factors SAM-pointed domain-containing Ets-like factor (SPDEF) and forkhead box protein A2 (FOXA2) regulate goblet cell differentiation. This study aimed to (1) investigate DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation and (2) compare this in airway epithelial cells from patients with COPD and controls during mucociliary differentiation. METHODS: To assess DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation, primary airway epithelial cells, isolated from trachea (non-COPD controls) and bronchial tissue (patients with COPD), were differentiated by culture at the air-liquid interface (ALI) in the presence of cytokine interleukin (IL)-13 to promote goblet cell differentiation. RESULTS: We found that SPDEF expression was induced during goblet cell differentiation, while FOXA2 expression was decreased. Importantly, CpG number 8 in the SPDEF promoter was hypermethylated upon differentiation, whereas DNA methylation of FOXA2 promoter was not changed. In the absence of IL-13, COPD-derived ALI-cultured cells displayed higher SPDEF expression than control-derived ALI cultures, whereas no difference was found for FOXA2 expression. This was accompanied with hypomethylation of CpG number 6 in the SPDEF promoter and also hypomethylation of CpG numbers 10 and 11 in the FOXA2 promoter. CONCLUSIONS: These findings suggest that aberrant DNA methylation of SPDEF and FOXA2 is one of the factors underlying mucus hypersecretion in COPD, opening new avenues for epigenetic-based inhibition of mucus hypersecretion.

Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear. METHODS: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain. RESULTS: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival. CONCLUSION: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression.

Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma. METHODS: Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2'-deoxycytidine (5-AZAC) was used to induce EpCAM expression. RESULTS: Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe. CONCLUSION: This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.

Secretion of thymidine kinase to increase the effectivity of suicide gene therapy results in the loss of enzymatic activity.

Low efficiency of gene transfer is one of the major limitations of gene therapy. A solution to this problem may be transmission; by modification of the transgene, the gene product can be secreted and internalized by the surrounding cells. Cancer gene therapy using the herpes simplex thymidine kinase (HSV-TK) suicide gene is a promising treatment, and TK has been used in clinical trials with some success. However, this kind of therapy has limited efficacy due to the low level of gene transfer reached. A modified TK protein, capable of migrating from the producing cell to neighboring cells, would result in a greater proportion of cells affected by the treatment. As a first step towards transmission, we constructed a secretory form of HSV-TK by including the Igkappa leader peptide in the gene. An endoplasmatic reticulum export signal was added to the construct to further improve its secretion. Secretion and protein production in cancer cells, the enzymatic activity of the modified proteins and the ability of the modified TK to sensitize cancer cells to ganciclovir were tested. Addition of the Igkappa leader resulted in high levels of secretion of HSV-TK, with up to 70% of the total amount of protein secreted. Inclusion of an ER export signal did not further improve secretion. The enzyme activity of the secreted TK however, was decreased when compared to native TK. This study is the first to report on secretion of TK, and provides a first step in a novel strategy to improve the efficiency of cancer gene therapy. The loss of function in secreted TK however, may present a major hurdle in the development of a transmitted form of TK.

A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer.

Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical cancer or premalignant cervical disease, as induced by high-risk type human papillomaviruses (HPVs). We previously demonstrated that immunization of mice with recombinant replication-defective Semliki Forest virus (rSFV) encoding a fusion protein of HPV16 E6 and -E7 (SFV-eE6,7) induces strong cytotoxic T-lymphocyte (CTL) activity and eradication of established HPV-transformed tumours. In this study, we compared the antitumour efficacy of SFV-eE6,7 with that of a recombinant adenovirus (rAd) type 5 vector, expressing the same antigen construct (Ad-eE6,7). Prime-boosting with SFV-eE6,7 resulted in higher precursor CTL frequencies and CTL activity compared to prime-boosting with Ad-eE6,7 and also in murine tumour treatment experiments SFV-eE6,7 was more effective than Ad-eE6,7. To elicit a therapeutic effect with Ad-eE6,7, 100/1000-fold higher doses were needed compared to SFV-eE6,7. In vivo T-cell depletion experiments demonstrated that these differences could not be explained by the induction of a different type of effector cells, since CD8+ T cells were the main effector cells involved in the protection against tumour growth in both rSFV- and rAd-immunized mice. Also comparable amounts of in vivo transgene expression were found upon immunization with rSFV and rAd encoding the reportor gene luciferase. However, anti-vector responses induced by a single injection with rAd resulted in a more than 3-log decrease in luciferase expression after a second injection of rAd. With rSFV, transgene expression was inhibited by only one to two orders of magnitude in preinjected mice. As an antigen-specific booster immunization strongly increases the level of the CTL response and is essential for efficient induction of immunological memory, it is likely that (part of) the difference in efficacy between rSFV and rAd type 5 can be ascribed to a diminished efficacy of the booster immunization in the case of rAd due to anti-vector antibody responses.

Nuclear imaging of hormonal receptor status in breast cancer: a tool for guiding endocrine treatment and drug development.

Breast cancer is a commonly occurring disease in women and a major cause of morbidity and mortality. In the past decades, the development of medical endocrine therapies has led to a significant improvement in treatment outcome for this type of cancer. This therapy is targeting specific hormone receptors that are overexpressed by the tumor cells. In breast cancer, estrogen and progesterone receptors are important targets and therefore the receptor status of the tumor strongly determines treatment outcome. However, the receptor status can change during the course of the disease and consequently therapy resistance can occur. Therefore, insight in the current receptor status of the tumor is essential for optimal treatment. Nuclear imaging techniques like positron emission tomography (PET) and single photon emission computed tomography (SPECT), could provide the means to monitor the receptor status of tumors and the receptor occupancy by medical endocrine drugs in a non-invasive manner. Thus, these imaging techniques could offer a tool to guide therapy management in the individual patient. Nuclear imaging techniques for some of the relevant receptors for treatment of breast cancer are currently available. These imaging techniques could also aid the development of novel treatment strategies like modulation of hormone receptor expression. This review will address the role of hormone receptors in breast cancer treatment, the available nuclear imaging methods for monitoring the receptor status, the potential role of nuclear imaging in therapy management and drug development.

Fusion of herpes simplex virus thymidine kinase to VP22 does not result in intercellular trafficking of the protein.

Suicide gene therapy is a promising approach for the treatment of cancer. Current protocols, however, suffer from low efficiency. We tried to alleviate this problem by developing a transgene that will spread from the initially transduced cell to the surrounding cells (transmission). We used herpes simplex virus (HSV) VP22 as a signal for cellular uptake of HSV-1 thymidine kinase (TK). By co-culturing naive cells with cells producing a TK-VP22 fusion protein, we detected intercellular trafficking of this protein. We used a variety of techniques, including two-color flow cytometry and cytotoxicity assays to detect the presence of TK in the non-producing cells. We confirmed intercellular migration of VP22. We did not detect any intercellular trafficking of the TK-VP22 fusion protein, by various fixation methods or flow cytometry. In ganciclovir sensitivity assays, we found no difference between the efficiency of TK (IC50=3.15+/-0.76 microg/ml) and TK-VP22 (IC50=2.27+/-0.59 microg/ml). Using a cell-free enzyme activity assay we showed that fusion of TK to VP22 did not change the enzyme activity. In conclusion, we described novel and robust methods to detect intercellular trafficking. From our data we concluded that protein transmission of TK by VP22 for gene therapy is not likely to be successful. In addition, we described a useful and quantifiable assay to measure the enzymatic activity of TK and TK fusion proteins, and described some common properties of VP22 fusion proteins that may explain the different results that have been obtained by others.

Highly efficient and carcinoma-specific adenoviral replication restricted by the EGP-2 promoter.

Although some successes have been reported using adenoviral vectors for the treatment of cancer, adenoviral cancer gene therapy is still hampered by the lack of sufficient tumor cell killing. To increase the efficiency, adenoviruses have been modified to replicate specifically in tumor tissues by using tumor specific promoters controlling genes essential for adenoviral replication. However, many conditionally replicating adenoviral vectors replicate in one tumor type only, which limits their application. The epithelial glycoprotein-2 (EGP-2) promoter is active in a broad variety of carcinomas, the most common type of cancer. We utilized this promoter to restrict adenoviral replication. In this report we demonstrate that the potency of the replication-competent adenovirus AdEGP-2-E1 to specifically lyse EGP-2 positive cells is comparable to wild-type adenovirus (AdWT). In addition, we show that in vivo AdEGP-2-E1 replicates as efficient as AdWT in EGP-2 positive tumor cells. On the contrary, in EGP-2 negative cell lines as well as in primary human liver samples, the replication was attenuated up to 4-log in comparison to wild-type virus. This report clearly shows the potency of the EGP-2 promoter to mediate highly efficient and specific adenoviral replication for carcinoma gene therapy.

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context.

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.

Enhanced transduction of fibroblasts in transplanted kidney with an adenovirus having an RGD motif in the HI loop.

Application of gene therapy to the renal graft has a powerful potential to improve the outcome of kidney transplantation and eliminate detrimental side effects associated with systemic therapy, through local expression of immunoregulatory or other protective molecules. However, the search for the optimal vector is still ongoing. In this study, we used a modified adenovirus that has an Arg-Gly-Asp (RGD) motif inserted in the HI loop of the fiber knob, as a successful strategy to transduce the renal graft. Donor Lewis rat kidneys were infused via the renal artery with a solution containing either a fiber-modified adenovirus (AdTL-RGD) or an unmodified adenovirus (AdTL), or with saline. Syngeneic recipients were killed after 3, 7 or 14 days. Efficiency, selectivity, localization, time course of gene expression and side effects were studied using biochemical and immunohistological techniques. Enhanced gene expression was achieved selectively in the transplanted kidney by AdTL-RGD, when compared to AdTL. Transgene expression lasted for at least 2 weeks. With the AdTL-RGD vector, the transgene was abundantly expressed in the renal interstitial fibroblasts. An increase in the number of cytotoxic T lymphocytes accompanied the use of either vector, when compared to saline. These data convincingly show enhanced and selective gene transfer to the fibroblasts of transplanted kidneys using an RGD-modified adenovirus, providing a highly efficient vector system for future therapeutic interventions.

An ex vivo human model system to evaluate specificity of replicating and non-replicating gene therapy agents.

BACKGROUND: Inefficiency, aspecificity and toxicity of gene transfer vectors hamper gene therapy from showing its full potential. On this basis significant research currently focuses on developing vectors with improved infection and/or expression profiles. Screening assays with validity to the clinical context to determine improved characteristics of such agents are not readily available since this requires a close relationship to the human situation. We present a clinically relevant tissue slice technology to preclinically test improved vector characteristics. METHODS: Slices were prepared from rat, mouse and human liver samples and from tumor tissue. Specificity of gene expression and replication was determined by infecting target and non-target tissue slices with transcriptionally retargeted adenoviruses and oncolytic viruses. RESULTS: Using rat liver slices, we demonstrate efficient knob-mediated adenoviral infectivity. A favorable tumor-on/liver-off profile, resembling in vitro and mouse in vivo data, was shown for a tumor-specific transcriptionally retargeted adenovirus by infecting slices prepared from tumor or liver tissue. Similar liver-off data were found for mouse, rat and human samples (over 3-log lower activity of the tumor-specific promoter compared to cytomegalovirus (CMV)). More importantly, we show that this technology when applied to human livers is a powerful tool to determine aspecific replication of oncolytic viruses in liver tissue. A 2- to 6-log reduction in viral replication was observed for a tumor-specific oncolytic virus compared to the wild-type adenovirus. CONCLUSIONS: The precision-cut tissue slice technology is a powerful method to test specificity and efficiency of gene transfer as well as of viral replication using human tissue.

Effects and interaction of 7-hydroxy methotrexate and methotrexate in leukaemic cells ex vivo measured by the thymidylate synthase inhibition assay.

In high dose therapy with methotrexate (MTX) the main metabolite 7-hydroxy-methotrexate (7-OH MTX) exceeds the plasma concentration of MTX achieving about tenfold higher levels. To investigate the interaction between 7-OH MTX and MTX ex vivo, the thymidylate synthase inhibition assay was used to quantify antifolate effects in patient blast samples, measuring the inhibition of the key enzyme thymidylate synthase (TS). In 18 leukemic samples (7 ALL, 11 AML) no dose-dependent TS inhibition was observed for 7-OH MTX. However, a statistically significant increase of TS inhibition (p<0.05) was observed for a 1:1 mixture of MTX and 7-OH MTX as compared to the effect of MTX alone. The half-maximal inhibitory concentrations in the short-exposure assay were 0.857 microM for MTX alone versus 0.088 microM for the 1:1 mixture with 7-OH MTX, respectively (p< or =0.05). This interaction was not observed with an excess of 7-OH MTX. Similar results were obtained in long exposure experiments. We conclude that there is a dose-dependent interaction between 7-OHMTX and MTX, despite the lack of TS inhibitory effects of the metabolite alone.

Protein transduction domains and their utility in gene therapy.

Protein transduction domains (PTDs, sometimes termed cell permeable proteins (CPP) or membrane translocating sequences (MTS)) are small peptides that are able to ferry much larger molecules into cells independent of classical endocytosis. This property makes PTDs ideal tools to transfer proteins and other molecules into living cells for research purposes. The mechanism by which this internalization takes place is poorly understood. It is evident, however, that many known PTDs bind to the same surface molecules (Heparan Sulphate Proteoglycans, HSPG) before internalization, and that internalization is dependent on these molecules. PTDs, although at this moment mainly used for the chemical or bacterial production of membrane permeable proteins can become powerful tools for gene therapy. By incorporating a PTD in the therapeutic gene product, the protein produced in the transfected cell might be enabled to spread to non-transfected cells, thereby creating an increased therapeutic effect. In this review, we give an overview of PTDs that may be useful for gene therapy applications, and discuss some of the problems that can be expected when incorporating PTDs in gene therapy approaches.

Targeted cancer gene therapy: the flexibility of adenoviral gene therapy vectors.

Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer efficiency on a wide spectrum of both dividing and non-dividing cell types. However, this broad tropism at the same time represents an important limitation for their use in therapeutic applications where specific gene transfer is required. This limitation may be overcome by using targeting approaches. In this regard, targeting may be achieved at three levels: transductional targeting, translational targeting and targeting of the expressed transgene. Here we describe our research efforts towards cancer specific gene therapy using these different targeting approaches. The results show that targeting of adenoviral vectors may be achieved using cancer specific cell surface molecules for transductional and transgene targeting or cancer specific promoters for transcriptional targeting. Combinations of these targeting approaches should result in optimized cancer specific gene therapy.

Expression of deoxycytidine kinase in leukaemic cells compared with solid tumour cell lines, liver metastases and normal liver.

Deoxycytidine kinase (dCK) is required for the phosphorylation of several deoxyribonucleoside analogues that are widely employed as chemotherapeutic agents. Examples include cytosine arabinoside (Ara-C) and 2-chlorodeoxyadenosine (CdA) in the treatment of acute myeloid leukaemia (AML) and gemcitabine to treat solid tumours. In this study, expression of dCK mRNA was measured by a competitive template reverse transcriptase polymerase chain reaction (CT RT-PCR) in seven cell lines of different histological origin, 16 childhood and adult AML samples, 10 human liver samples and 11 human liver metastases of colorectal cancer origin. The enzyme activity and protein expression levels of dCK in the cell lines were closely related to the mRNA expression levels (r=0.75, P=0.026 and r=0.86, P=0.007). In AML samples, dCK mRNA expression ranged from 1.16 to 35.25 (x10(-3)xdCK/beta-actin). In the cell line panel, the range was 2.97-56.9 (x10(-3)xdCK/beta-actin) of dCK mRNA expression. The enzyme activity in liver metastases was correlated to dCK mRNA expression (r=0.497, P=0.05). In the liver samples, these were not correlated. dCK mRNA expression showed only a 36-fold range in liver while a 150-fold range was observed in the liver metastases. In addition, dCK activity and mean mRNA levels were 2.5-fold higher in the metastases than in the liver samples. Since dCK is associated with the sensitivity to deoxynucleoside analogues and because of the good correlation between the different dCK measurements in malignant cells and tumours, the CT-RT PCR assay will be useful in the selection of patients that can be treated with deoxycytidine analogues.