[Fluorescence diagnosis and photodynamic therapy with 5-aminolevulinic acid induced protoporphyrin IX in gynecology: an overview]. / Fluoreszenzdiagnostik und photodynamische Therapie mit 5-Aminolävulinsäure induziertem Protoporphyrin IX in der Gynäkologie: eine Ubersicht.

Fluorescence diagnosis and photodynamic therapy with 5-aminolevulinic acid induced protoporphyrin IX are promising new options in the diagnosis and therapy of diseases in a wide spectrum of medical disciplines such as urology, dermatology, gastroenterology, surgery, neurosurgery and gynecology. The techniques are based on the application of the heme biomolecule precursor 5-aminolevulinic acid which induces the endogenous accumulation of the photosensitizer protoporphyrin IX in designated tissues. After external excitation with blue light a strong red fluorescence can be initiated particularly in tumorous tissues. In gynecology many studies have been performed evaluating the usefulness of fluorescence based detection of cervical dysplasias, breast cancer, endometrial diseases, ovarian cancer and endometriosis. This work aims on the principles of fluorescence detection as an important tool in biomedical optical imaging and its current status in gynecology.

Clinical multi-colour fluorescence imaging of malignant tumours--initial experience.

PURPOSE: The detection of malignant tumours relies on a variety of diagnostic procedures including X-ray images and, for hollow organs, endoscopy. The purpose of this study was to present a new technique for non-invasive tumour detection based on tissue fluorescence imaging. MATERIAL AND METHODS: A clinically adapted multi-colour fluorescence system was employed in the real-time imaging of malignant tumours of the skin, breast, head and neck region, and urinary bladder. Tumour detection was based on the contrast displayed in fluorescence between normal and malignant tissue, related to the selective uptake of tumour-marking agents, such as haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), and natural chromophore differences between various tissues. In order to demarcate basal cell carcinomas of the skin, ALA was applied topically 4-6 h before the fluorescence investigation. For urinary bladder tumour visualisation (transitional cell carcinoma of different stages including carcinoma in situ), ALA was instilled into the bladder 1-2 h prior to the study. Malignant and premalignant lesions in the head and neck region were imaged after i.v. injection of HPD (Photofrin). Finally, the extent of in situ and invasive carcinomas of the breast was investigated in surgically excised specimens from patients that received a low-dose injection of HPD 24 h prior to the study. The tumour imaging system was coupled to an endoscope. Fluorescence light emission from the tissue surface was induced with 100-ns-long optical pulses at 390 nm, generated from a frequency-doubled alexandrite laser. With the use of special image-splitting optics, the tumour fluorescence, intensified in a micro-channel plate, was imaged in 3 selected wavelength bands. These 3 images were processed together to form a new optimised-contrast image of the tumour. This image, updated at a rate of about 3 frames/s, was mixed with a normal colour video image of the tissue. RESULTS: A clear demarcation from normal surrounding tissue was found during in vivo measurements of superficial bladder carcinoma, basal cell carcinoma of the skin, and leukoplakia with dysplasia of the lip, and in in vitro investigations of resected breast cancer. CONCLUSIONS: The initial clinical experience of using multi-colour fluorescence imaging has shown that the technique has the potential to reveal malignant tumour tissue, including non-invasive early carcinoma and also precancerous tissue. Further investigations are needed to fully develop the method.

[Lipid metabolism in retinal pigment epithelium. Possible significance of lipoprotein receptors]. / Lipidmetabolismus im Retinalen Pigmentepithel (RPE). Mögliche Bedeutung von Lipoprotein-Rezeptoren.

PURPOSE: The retinal pigment epithelium (RPE) regulates the lipid metabolism of the photoreceptors by catalysis of membrane outer segments and via choriocapillary perfusion is also exposed to the regulation of blood lipid levels. Since the uptake the metabolism of cholesterol are mediated by specific low-density lipoprotein (LDL) receptors, expression and regulation of this receptor-type were studied in RPE cultures. METHODS: In vitro experiments were carried out in transformed (SV40) human RPE cells. Human fibroblasts were used as a comparative cell line with known receptor expression. LDL was coupled to a fluorescent marker (Dil); receptor binding was quantified by flow cytometry. Expression and saturation characteristics were determined. LDL metabolism was examined by variation of the temperature (4 and 37 degrees C). LDL and Dil-LDL showed competition at the receptor. RESULTS: RPE cells demonstrated a higher uptake of Dil-LDL than fibroblasts. Expression could be further stimulated by culture conditions. Uptake kinetics were saturable with complete saturation at 50 micrograms/ml Dil-LDL. LDL uptake was shown to be temperature-dependent, indicating an energy-dependent pathway. CONCLUSIONS: RPE cells exhibit significant expression of receptors for native LDL, possibly mediating the lipid metabolism of the RPE-photoreceptor complex, as well as the uptake of blood lipids. Lack of regulation of the receptor for LDL may lead to intracellular accumulation of lipids, which may play a role in the pathogenesis of AMD.

Photodynamic targeting of human retinoblastoma cells using covalent low-density lipoprotein conjugates.

Combination of photosensitizers with carrier molecules has been shown to enhance the efficiency of photodynamic therapy (PDT). Owing to an increased expression of their receptors on some malignant and proliferating cells, low-density lipoproteins (LDLs) are potential endogenous carriers. A photosensitizer, chlorin e6 (Ce6), was covalently bound to LDL via carbodiimide activation. The Ce6-LDL conjugate was evaluated on a fibroblast cell line with defined LDL receptor expression and a retinoblastoma cell line (Y79). Uptake of free Ce6 and Ce6 either covalently bound to or complexed with LDL was measured by spectrofluorimetry. Phototoxicity after irradiation at 660 nm was determined by a mitochondrial activity assay (MTT). Covalent binding to LDL significantly increased the uptake of Ce6 for both cell lines by a factor of 4-5. A Ce6: LDL binding ratio of 50:1 was optimal. A receptor-mediated uptake was demonstrated by saturability and competitive inhibition by free LDL. Binding also occurred at 2 degrees C and was attributed to non-specific associations. Irradiation with 10 J cm-2 of 660 nm light after treatment of cells with Ce6-LDL conjugate reduced the MTT activity by 80%, while free or mixed Ce6 induced a maximum of 10% reduction in the MTT activity following identical treatment conditions. These data suggest that targeting of LDL receptor-bearing cells using covalently bound carriers, such as LDL, might increase the efficiency and selectivity of PDT. Intraocular tumours such as retinoblastomas could be appropriate targets for such an approach owing to the ease of access of light sources and the need for non-invasive approaches in sensitive ocular sites.

Photodynamic laser therapy for rheumatoid arthritis. Cell culture studies and animal experiments.

The introduction of arthroscopic techniques has improved the surgical therapy of rheumatoid arthritis. The additional application of the holmium:yttrium aluminum garnet (Ho:YAG) laser likewise holds great promise by providing complete hemorrhagic control. Unfortunately, a minimally invasive solution for use in smaller joints has not yet emerged. The present study describes the possible treatment of these joints by means of photodynamic laser therapy. Cell culture studies with human synovial fibroblasts obtained from patients with rheumatoid arthritis have demonstrated a cytotoxic effect after administration of Photosan-3 as a photosensitizer and subsequent laser irradiation at 630 nm. for the in vivo studies, IgG-induced arthritis in rabbits, which is histologically consistent with the proliferative phase of rheumatoid arthritis, was used as the animal model. The histologic picture following photodynamic laser therapy with Photosan-3 revealed complete synovial destruction which also extended to the border of the subjacent joint capsule. In contrast, bradytrophic structures, e.g. cartilage. menisci, and ligaments, remained unchanged at both the macroscopic and microscopic levels. Therefore, photodynamic laser therapy can be considered a new method in the surgical treatment of inflammatory disease of the synovial membrane. It has the advantage of being minimally invasive, while offering a high degree of efficacy and selectivity.

Chemical instability of 5-aminolevulinic acid used in the fluorescence diagnosis of bladder tumours.

Aqueous solutions of 5-aminolevulinic acid (ALA) prepared for intravesical instillation in the framework of a clinical study on the fluorescence diagnosis of urothelial bladder cancer were found to be unstable. This chemical instability of ALA was studied in aqueous solution of 37 degrees C as a function of concentration, pH and reaction time. Our investigations showed that the reaction of ALA is an irreversible process, which yields at least two reaction products in the pH range studied (pH lower than 8): the 2,5-(beta-carboxyethyl)dihydropyrazine and the 2,5-(beta-carboxyethyl)pyrazine. As a result of these studies, the conditions for the preparation of ALA solutions to be used for intravesical instillation were optimized: solution of ALA in phosphate-buffered saline at a concentration of 0.18 M (3 g of ALA in 100 ml) neutralized to pH 5, were prepared and stored on ice until use. Solutions prepared under these conditions were stable and were used for fluorescence diagnosis of bladder tumours with successful results. The effect of the pH and the composition of the urine on the extent of the reaction of ALA and on the nature of its reaction products formed during instillation was investigated by comparing the urine of patients before and immediately after instillation of ALA.

Spectral characterization of the benzoporphyrin derivative monoacid ring-A photoproduct formed in fetal calf solutions during irradiation with 694 nm continuous-wave radiation.

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a second-generation photosensitizer for photodynamic therapy (PDT) that has shown good results in phase I clinical trials. Similar to other porphyrin derivatives, BPD-MA readily photobleaches during in-vivo PDT treatment. This study investigated the photodegradation of BPD-MA in fetal calf serum (FCS) solutions in vitro. Absorption and fluorescence spectra from dilute solutions of BPD-MA in 10% FCS were recorded before and immediately after irradiation with light at 694 nm. After irradiation, the appearance of a new fluorescence emission band at 650 nm and changes in the fluorescence excitation spectra indicate the formation of a photoproduct. Photoproduct formation was observed only when BPD-MA was bound to FCS and in oxygenated solutions. The spectroscopy of the photoproduct is consistent with the reaction of an oxygen species with the ring B vinyl group, forming a hydroxyaldehyde photoproduct. Monitoring the increase in photoproduct fluorescence during treatment may provide an in-vivo dosimeter to measure PDT efficacy.

[Experimental bases for photodynamic laser therapy in chronic polyarthritis]. / Experimentelle Grundlagen einer photodynamischen Lasertherapie für die chronische Polyarthritis.

The principle of photodynamic laser therapy (PDT) for chronic polyarthritis consists in specifically concentrating a drug (photosensitizer) in the synovium. Subsequent activation of the photosensitizer by means of laser leads to a cytotoxic effect. The practicability of PDT was first shown in cell cultures of human synovial fibroblasts. For further tests an animal model consisting of IgG-induced arthritis in rabbits was used. In this model, concentration of the photosensitizer in the synovial lining cells, in the media of arteries and in the lymphoid infiltrate was seen. After laser irradiation there was total selective demarcation of the synovium. In contrast, bradytrophic tissues such as cartilage, meniscus and ligament structures were changed neither macroscopically nor microscopically. In the animal model PDT combines high selectivity with minimal invasiveness and can be used in small joints. PDT thus offers ideal preconditions for future minimal invasive treatment of chronic inflammatory joint diseases.

Beta-tubulin and P-glycoprotein: major determinants of vincristine accumulation in B-CLL cells.

Vincristine (VCR) accumulation in chronic lymphatic leukemia of B-cell origin (B-CLL) has recently been shown not to be inversely correlated to P-glycoprotein (PGP) levels. Therefore, we studied, in addition to PGP expression and accumulation of VCR, the cellular beta-tubulin content in quiescent and rhIL-2 activated B-CLL cells. VCR mediates cytotoxicity by binding to tubulin. Constitutive beta-tubulin levels in B-CLL cells varied considerably. Upon activation with rhIL-2, beta-tubulin expression increased significantly. Therefore, tubulin levels could be correlated over a wide range to VCR accumulation. When the PGP-mediated drug efflux was blocked by verapamil (VRP), tubulin levels correlated linearly to VCR accumulation. All B-CLL cases expressed PGP at different levels. There was no linear correlation between PGP expression and VCR accumulation. A modulation factor m was defined as a quotient of VCR accumulation in the presence and absence of VRP to define the extent by which VRP inhibited a steady-state accumulation of VCR. The factor allowed discrimination between B-CLLs expressing low versus high PGP, irrespective of the levels of tubulin. However, PGP and beta-tubulin levels together were predictive for VCR accumulation in steady state. There was no uniform-accumulation defect for VCR in B-cell CLL because beta-tubulin and PGP were expressed independently. Non PGP-mediated VCR transport seems to play a minor role in B-cell CLL. Leukemia-associated varying of cytoskeletal organization in B-cell CLL might be one reason for the diverse cellular responses to receptor-mediated signals.

[Photodynamic therapy of eyelid basalioma after topical administration of delta-aminolevulinic acid]. / Die Photodynamische Therapie (PDT) des Lidbasalioms nach topischer Applikation von delta-Aminolävulinsäure (ALA).

Delta-aminolevulinic acid (ALA) induced Protoporphyring IX photodynamic therapy (PDT) is a new method in the therapy of cutaneous malignancies. Topical application prevents photosensibilization of normal skin. Promising results in the treatment of superficial basal cell carcinomas have been reported. To evaluate the efficiency of this method in the therapy of basal cell carcinomas of the lid, 10 patients were treated by PDT 5 h after application of ALA. The lesions were irradiated by an argon-ion-laser-pumped dye laser at a wavelength of 630 or 635 nm and at cumulative radiant exposures of 50 and 100 J/cm2. After 5-8 weeks follow-up, the basal cell carcinomas were excised to assess histologic changes. In the first few days after PDT the lesions seemed clinically to regress, due to erythematous swelling, but after a few weeks all tumours showed the same configuration they had before irradiation. Histological examination revealed small areas of necrotic cells in all irradiated basal cell carcinomas, surrounded by residual tumour formations. The reason for the poor results may be limited penetration of ALA or light. Although ALA-induced Protoporphyrin-IX PDT is a promising approach in the therapy of dermal lesions, it is not yet an acceptable alternative method in the treatment of basal cell carcinomas of the lid.

Chemomodulation of drugs involved in multidrug resistance in chronic lymphatic leukemia of the B-cell type.

Reduced drug accumulation may be one reason for intrinsic drug resistance in chronic lymphatic leukemia of the B-cell type (B-CLL). Immunophenotyped leukemic human B-cells from 38 cases of B-CLL were characterized for P-glycoprotein (PGP) content. In all, 30 cases of B-CLL were additionally analyzed for further parameters: accumulation of daunorubicin (DNR, n = 20) and rhodamine 123 (Rh123, n = 30) in both the presence and the absence of verapamil (VRP). Also, 16 cases of B-CLL were analyzed for vincristine (VCR) accumulation with or without VRP. Concerning the relative expression of PGP, these 16 cases of B-CLL were representative for the whole group of 30 cases. Spontaneous accumulation of Rh123 and VCR varied over a wide range: accumulation of Rh123, by a factor of 11.8; accumulation of VCR, by a factor of 9.7; and accumulation of DNR, by a factor of 3.6. VRP modulated the accumulation of RH123 in 16/30 cases (53%), that of VCR in 69% of cases, and that of DNR in 11% of cases. The maximal VRP-mediated increases in accumulation amounted to factors of 1.3 for DNR, 2.3 for Rh123, and 7.8 for VCR. Spontaneous drug accumulation did not correlate with the extent of chemomodulation. The amount of PGP in B-CLL cells (all cases studied were PGP-positive) did not correlate with drug accumulation or with the extent of VRP-mediated chemomodulation. Thus, high expression of PGP is only partially responsible for defective drug accumulation in B-CLL. Only the degree of chemomodulation by VRP is predictive for the extent of the PGP-related functional drug accumulation defect. Thus, in B-CLL, PGP-independent drug accumulation defects seem to be as important as those mediated by PGP. The extent of this drug accumulation defect varies for the different drugs in the following order VCR > Rh123 > DNR. The relevance of PGP-mediated and -independent drug accumulation defects in vivo may depend to a large extent on the drug being used and on the individual cell type. Both types of defect in drug accumulation are of high importance when regimens include VCR a drug commonly used in second-line chemotherapy of B-CLL. Both defects in drug accumulation may be responsible for intrinsic VCR resistance in B-CLL.

MDR hamster cells exhibiting multiple altered gene expression: effects of dexniguldipine-HCl (B859-35), cyclosporin A and buthionine sulfoximine.

An actinomycin D selected, multidrug-resistant (MDR) hamster CHO subline showed strong expression of the P-glycoprotein and sorcin genes together with several other alterations such as a: (i) reduced growth rate, (ii) lowered topoisomerase II, (iii) lowered glutathione-S-transferase-P gene expression, and (iv) the emergence of a 15.5 kDa protein. Besides high resistances to adriamycin, actinomycin D, and vincristine, we observed a lowered sensitivity towards bleomycin, a rather hydrophilic drug usually not involved in P-glycoprotein associated MDR. Moreover, the MDR subline showed a pronounced collateral (enhanced) sensitivity towards the sterically pure dihydropyridine anticancer drug dexniguldipine-HCl (B859-35) preventing its characterization for MDR modulation here. At a non-cytotoxic dose (10 microM) the immunosuppressive cyclic peptide cyclosporin A completely abolished the resistance to vincristine, partially reversed the resistance to teniposide and strongly enhanced the sensitivity towards bleomycin, while not influencing the drug sensitivities of the parental cell line. Buthionine sulfoximine (BSO), an agent depleting cellular glutathione levels, distinctly increased the sensitivity towards teniposide at nontoxic doses (50 microM) exclusively in the MDR subline, while it did not alter vincristine or bleomycin cytotoxicity.

Mdr1/P-glycoprotein, topoisomerase, and glutathione-S-transferase pi gene expression in primary and relapsed state adult and childhood leukaemias.

In a variety of adult and childhood leukaemia cell samples collected at different states of the disease, we analysed in a series of sequentially performed slot-blot or Northern-blot hybridisation experiments the expression of genes possibly involved in multiple drug resistance (MDR) (mdr1/P-glycoprotein, DNA topoisomerase II, glutathione-S-transferase pi), and the expression of the DNA topoisomerase I and histone 3.1 genes. Occasionally, P-glycoprotein gene expression was additionally examined by indirect immunocytofluorescence using the monoclonal antibody C219. No significant difference in mdr1/P-glycoprotein mRNA levels between primary and relapsed state acute lymphocytic leukaemias (ALL) was seen on average. Second or third relapses, however, showed a distinct tendency to an elevated expression of this multidrug transporter gene (up to 10-fold) in part well beyond the value seen in the moderately cross-resistant T-lymphoblastoid CCRF-CEM subline CCRF VCR 100. Increased mdr1/P-glycoprotein mRNA levels were also found in relapsed state acute myelogenous leukaemias (AML), and in chronic lymphocytic leukaemias (CLL) treated with chlorambucil and/or prednisone for several years. Topoisomerase I and topoisomerase II mRNA levels were found to be very variable. Whereas in all but one case of CLL topoisomerase II mRNA was not detected by slot-blot hybridizations, strong topoisomerase I and topoisomerase II gene expression levels, frequently exceeding the levels monitored in the CCRF-CEM cell line, were seen in many cell samples of acute leukaemia. If topoisomerase II mRNA was undetectable, expression of topoisomerase I was clearly visible throughout. These observations might be valuable considering the possible treatment with specific topoisomerase I or topoisomerase II inhibitors. Significant positive correlations were found (i) for topoisomerase I and histone 3.1 gene expression levels in general (P less than 0.001), and (ii) in the CLL samples additionally for the expression levels of the mdr1 gene, and the histone 3.1, topoisomerase I, and glutathione-S-transferase pi genes, respectively.

Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells.

Considering the possibility to overcome drug resistance by other treatment strategies than chemotherapy we investigated the susceptibility of three independently selected multidrug-resistant sublines of the T-lymphoblastoid leukemic cell line CCRF-CEM to lymphokine-activated killer (LAK) cells. We found that two of the multidrug-resistant sublines were significantly less susceptible targets to LAK cells. A third one, however, was as susceptible as the parental CCRF-CEM cell line. Moreover, a multidrug-resistant subline that reverted to an almost drug-sensitive phenotype was observed to be also revertant for resistance against LAK cells. We found an inverse relationship between the expression of the mdr1 gene (P-glycoprotein) and the susceptibility to LAK cells. Verapamil, a calcium channel blocker, while increasing the drug sensitivity of a multidrug-resistant subline, did not induce a reversal of the suppression of LAK susceptibility. The possibility of enhanced resistance to LAK cells of multidrug-resistant cells should be taken into account when one is looking for therapy strategies to overcome multidrug resistance.

Changes in clonal growth, immunophenotype, and morphology during a follow-up study of an acute lymphoblastic leukemia.

Cells of a 21-year-old patient with acute lymphatic leukemia were analyzed for morphology and immunophenotype and for genotype consecutively during the course of disease. Initial therapy with the BMFT-ALL protocol (Bundesministerium für Forschung und Technologie) reduced leukemic cells only marginally. The following high-dose Ara-C, mitoxantrone (HAM) chemotherapy led to a cell reduction of 75% and to a drastic change in cell morphology from initially 90% blasts to mainly small lymphoid cells. Immunophenotype, which showed 90% CD7-positive cells in the beginning with a prevalence of helper (60%) over suppressor cells (15%) remained fairly constant until the onset of HAM chemotherapy, which led to a sharp fall and a subsequent slow increase in all T-cell markers. In contrast to pretherapeutic findings, CD7 was now only expressed on the small cells and not on blast cells. Southern blot analysis of the T-cell receptor configuration revealed an initially monoclonal population with rearranged T beta gene. A new band appearing during the clinically ineffective therapy was indicative for development of a second small population which did, however, not emerge in immunophenotype analysis. This second population was eliminated by the HAM chemotherapy, leaving back the initial clone responsible for the final fatal outcome. No activity of the multidrug resistance gene could be detected by Northern blotting.

Resistance to methotrexate and multidrug resistance in childhood malignancies.

Resistance to drugs, either primary or acquired, is a main problem in cancer chemotherapy. The paper summarizes our results in regard to resistance to methotrexate and multiple drug resistance in human cell lines of pediatric malignancies and in children with resistant cancer. In cell lines as well as in children we could demonstrate amplification of the gene coding for dihydrofolate reductase as a cause for resistance to MTX. Procedures to overcome drug resistance such as treatment with high dose MTX and leucovorin rescue are discussed. The increased expression of the mdrl gene coding for the P-glycoprotein is related to multidrug resistance. This could be shown in cell lines and in children. The expression decreased when the drug, used for induction of resistance, was omitted for a few weeks from the cell culture medium. Readdition of the drug caused a rapid increase of expression. For the first time data in children are presented which demonstrate the amplification of the gene coding for dihydrofolate reductase or increased expression of the mdrl gene as cause of drug resistance. The clinical implications of these findings are discussed.

Metabolites of microorganisms. 248. Synthetic analogs of saphenamycin.

A synthesis of racemie saphenic acid is described. From this acid 9 ester derivatives of saphenamycin were prepared. Those with aromatic acid components showed high activity against many Gram-positive and some Gram-negative bacteria. Of the esters with aliphatic acid moieties only the acetate and, to a lesser extent, the butyrate showed considerable anti-bacterial activities, whereas esters with higher fatty acids showed strongly reduced, if any, activities against some test organisms. Similar results were obtained with ID50 values against the eucaryotic tumor cell line CCRF/CEM. The salicylate, which is structurally similar to saphenamycin, was most active.

Expression of a P-glycoprotein gene is inducible in a multidrug-resistant human leukemia cell line.

A human T lymphoblastoid CCRF-CEM cell line exhibiting cross resistance to a variety of drugs was selected with increasing doses of actinomycin D. A subline, designated CCRF ACTD400+, was permanently cultured in the presence of 400 ng/ml Actinomycin D for several months. Using a fragment of the human mdr1 cDNA we found high expression of a 5 kb mRNA species which was not detectable in the sensitive parental CCRF-CEM cell line. The extent of the mdr-mRNA expression in resistant cells, however, depended on the presence or absence of actinomycin D in the culture medium: when the inhibitor was omitted, the expression decreased to about 60% after one month. In reverse, the steady state level of the P-glycoprotein mRNA increased about 2.5-fold within 72 h after the original dose of the drug was added again. In further experiments we recorded the actinomycin D or adriamycin dose response curves of the variously treated sublines by evaluation of [3H]uridine or [3H]thymidine incorporation, respectively, into acid insoluble material. Consistently, the drug sensitivity of the respective macromolecular synthesis was found to decrease with increasing mdr-mRNA levels.

Characterization of actinomycin-D-resistant CHO cell lines exhibiting a multidrug-resistance phenotype and amplified DNA sequences.

Actinomycin D (DACT)-resistant sublines of the Chinese hamster ovary cell line CHO-K1 were selected in vitro. Sublines were derived which expressed 5.2-fold (CHO 15DACT) and 35.8-fold (CHO 100DACT) resistance to DACT. The CHO 100DACT subline displayed marked cross-resistance to bleomycin, adriamycin, daunomycin, vinblastine, vincristine, VP 16 and VM 26. No cross-resistance was found to cisplatin or methotrexate. The resistant cells exhibited enhanced (collateral) sensitivity to prednisolone. Combination of prednisolone with vincristine resulted in a pronounced synergistic effect on sensitive cells, whereas in resistant cells the combined effect of both drugs was merely additive. Resistant cells, viably stained with the DNA-specific dye Hoechst 33342, exhibited decreased fluorescence intensities compared to parental cells. In contrast to sensitive cells the resistant sublines did not accumulate the mitochondria-specific dye rhodamine 123. Co-incubation with verapamil, however, effectively enhanced accumulation of the dye. The potential diagnostic value of these fluorescent compounds as marker dyes for the multidrug-resistance phenotype is discussed. Non-toxic doses of verapamil almost completely reversed the resistance to various drugs in CHO 100DACT cells. Specific DNA sequences were amplified in resistant cells, and the increase in resistance was paralleled by a concomitant increase in the copy number of these sequences, suggesting that the corresponding gene may be functionally linked to the multidrug-resistance phenotype.