A study of diurnal proliferative activity in tumour and small intestine of C3H mice bearing a transplanted mammary carcinoma

Diurnal changes in proliferative activity were investigated in tumour and small intestinal epithelium of mice bearing a transplanted mammary carcinoma. In addition to mitotic and labelling index studies, the metaphase-arrest technique with vincristine (VCR) was employed. In the tumour there was no clear evidence of a significant diurnal rhythm in proliferative activity but in the small intestinal epithelium such a rhythm was clearly demonstrated. A higher cell production rate (kB) measured by metaphase-arrest and higher labelling and mitotic indices were seen in the mid to late part of the dark period. The peak mitotic index was seen 3 to 6 h after the labelling peak in the small intestine. The basal third of the crypt which is believed to include the stem cell compartment of this tissue showed larger diurnal fluctuations in both labelling index and kB than the rest of the proliferative compartment.     

ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells

To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.     

Multidrug resistant HOB1 lymphoma cells express P-glycoprotein that does not play the major role in the development of drug resistance to adriamycin.

Two cell lines resistant to 0.1 microM vincristine (VCR) and 2.0 microM adriamycin (ADR), respectively, (designated HOB1/VCR0.1 and HOB1/ADR2.0) were established from a human immunoblastic B lymphoma cell line. These cell lines showed the typical MDR phenotype with overexpression of P-glycoprotein and decreased [3H]VCR accumulation. The retention amounts of intracellular [3H]VCR in these two cell lines could be augmented by verapamil. However, in spite of the overproduction of P-glycoprotein, both HOB1/VCR1.0 and HOB1/ADR2.0 cells did not exhibit decreased accumulation of intracellular [14C]ADR. And the retention of [14C]ADR was not affected by verapamil. Our data support that P-glycoprotein is a drug transporter more important for the development of drug resistance to VCR than to ADR.     

Comparison of lymphocyte production in lymphoid organs and their compartments using the metaphase-arrest technique

Summary Lymphocyte proliferation was studied in normal young anesthetized pigs by the metaphase-arrest technique using vincristine (VCR). In each animal biopsies were taken simultaneously from the thymus, mesenteric lymph nodes, spleen, palatine tonsil and Peyer's patches from the ileum and jejunum. After taking the first samples, 0.25 mg VCR/ kg body weight was injected i.v. and then four more biopsies were excised for up to 3.5 h after VCR. Imprints of the lymphoid organs were evaluated as an overall index for each organ, and histological sections were used to determine the mitotic index in typical B-and T-lymphocyte areas in these organs. In follicles of mesenteric lymph nodes, tonsils and the two types of Peyer's patches a comparable increase in the mitotic index was found, 3.62% per hour. In the corona the increase was also comparable but much lower, 0.43% per hour and in the interfollicular area similarly 0.38% per hour. In the spleen the mitotic rate was 0.69% for the white pulp and 0.42% per hour for the red pulp. In the thymic cortex the mitotic index increased by 0.49% and in the medulla by a surprisingly high value of 0.32% per hour. The metaphase-arrest technique in larger animals enables a comparison of lymphocyte production among organs and their different compartments, and demonstrates the important contribution of peripheral lymphoid organs to the renewal of the lymphocyte pools.List of Abbreviations DNA deoxyribonucleic acid - ¹⁴ C-TdR thymidine labelled with ¹⁴C - ³ H-TdR tritiated thymidine - r _m rate of entry of cells in mitosis per hour - VCR vincristine sulphate Partly presented at the XII Int. Anat. Congress in London, August 1985     

AS602801 sensitizes glioma cells to temozolomide and vincristine by blocking gap junction communication between glioma cells and astrocytes

Previous studies showed that the chemotherapeutic effect of temozolomide (TMZ) and vincristine (VCR) against glioma might be blunted by the co-culture with astrocytes, and connexin-43 (CX43) was thought to play a vital role in the communication between glioma cells and astrocytes. In this study, we aimed to investigate the combined chemotherapeutic effect of AS602801 and TMZ/ VCR in glioma cells both. Dye transfer assay was used to evaluate the gap junction activity between U251 cells and astrocytes. Western blot and immunohistochemistry were carried out to analyse the expression of p-JNK, CX43 and CASP-3 proteins treated under different conditions. AS602801 significantly suppressed the gap junction activity between U251 cells and astrocytes. The expression of p-JNK and CX43 was remarkably inhibited by AS602801. TMZ/VCR-induced apoptosis of glioma cells was effectively enhanced by AS602801 treatment. Accordingly, the inhibitory role of TMZ/VCR in the expression of p-JNK, CX43 and CASP-3 in glioma cells was notably restored by AS602801. Furthermore, in a glioma cell xenograft, AS602801 showed an apparent capability to enhance TMZ/VCR-induced tumour cell apoptosis through altering the expression of p-JNK, CX43 and CASP-3. The findings of this study demonstrated that the co-culture of glioma cells with astrocytes blunted the tumour killing effect of TMZ and VCR. AS602801 down-regulated CX43 expression by inhibiting JNK. And AS602801 also sensitized glioma cells to TMZ/VCR by blocking the gap junction communication between glioma cells and astrocytes via down-regulating CX43, indicating its potential role as a novel adjuvant chemotherapeutic agent in the treatment of glioma.     

Comparative investigations on the effect of different dose schedules of the phase-specific drug vincristine (VCR) on the proliferation kinetics of a solid experimental tumour

The aim of the present investigation was to study the effect of a high bolus injection (1 X 2.1 mg) of vincristine (VCR) during the phase of tumour growth retardation at the 14th day after transplantation and to compare the findings with the results of single (1 X 0.35 mg) and repeated (6 X 0.35 mg) applications of this cytostatic drug. Furthermore, an attempt was made to induce a synchronization of tumour cell proliferation. It was found that the effect on the volume growth was very pronounced after the high bolus injection and the repeated application of vincristine compared with the single low dose of the cytostatic drug. A synchronization of the tumour cell proliferation by flow out of the mitotic block could not be demonstrated. On the other hand a modest simultaneous recruitment of previously non-cycling tumour cells into the cell cycle occurred in the periphery of the tumour after the high bolus injection. The repeated application and the high bolus injection of VCR increased the cytostatic effect, especially in the tumour centre, related to the more slowly proliferating tumour cell compartment.     

Comparative Investigations On the Effect of Different Dose Schedules of the Phase-Specific Drug Vincristine (Vcr) On the Proliferation Kinetics of A Solid Experimental Tumour

The aim of the present investigation was to study the effect of a high bolus injection (1 × 2.1 mg) of vincristine (VCR) during the phase of tumour growth retardation at the 14th day after transplantation and to compare the findings with the results of single (1 × 0.35 mg) and repeated (6 × 0.35 mg) applications of this cytostatic drug. Furthermore, an attempt was made to induce a synchronization of tumour cell proliferation. It was found that the effect on the volume growth was very pronounced after the high bolus injection and the repeated application of vincristine compared with the single low dose of the cytostatic drug. A synchronization of the tumour cell proliferation by flow out of the mitotic block could not be demonstrated. On the other hand a modest simultaneous recruitment of previously non-cycling tumour cells into the cell cycle occurred in the periphery of the tumour after the high bolus injection. the repeated application and the high bolus injection of VCR increased the cytostatic effect, especially in the tumour centre, related to the more slowly proliferating tumour cell compartment.     

A CRITICAL REVIEW OF THE USE OF VINCRISTINE (VCR) AS A TUMOUR CELL SYNCHRONIZING AGENT IN CANCER THERAPY

Vincristine (VCR) has been used clinically in so-called ‘tumour cell synchronization therapy schedules'. These schedules are based on the assumption that cells, arrested in metaphase by low doses of VCR, subsequently re-enter the proliferative cycle synchronously. However, the evidence that tumour cell synchrony can be achieved under clinical conditions or that ‘cell synchronization therapy schedules’ yield a better therapeutic response than other efficient combination schemes, is scanty. Further, even in experimental systems, the efficacy of VCR as a cell synchronizing agent is disputed. Indeed, in some systems, cells arrested in metaphase by low doses of VCR, do not re-enter a normal proliferative cycle at all following arrest. In addition, the complex nature of the VCR—tumour interaction and the heterogeneous nature of the tumour cell populations against which it is used augurs badly for the successful application of cell synchronization therapy schedules.     

AN IN VIVO DOUBLE LABELLING STUDY OF THE SUBSEQUENT FATE OF CELLS ARRESTED IN METAPHASE BY VINCRISTINE IN THE JB-1 MOUSE ASCITES TUMOUR

The fate of cells arrested by Vincristine (VCR) in metaphase is of interest because of the wide use of this substance in cancer chemotherapy and, particularly, in relation to its use in so-called ‘synchronization’ therapy. The present study was designed to answer the question of whether cells blocked in metaphase by VCR subsequently proliferate further or whether they become infertile and die. By means of a double labelling technique with [³H] and [¹⁴C]thymidine (TdR) it was shown that all VCR-arrested metaphases in the JB-1 ascites tumour subsequently became necrotic. These cells did not re-enter a viable G₂ phase following arrest and thus could not take part in a wave of synchronous proliferation. In agreement with earlier studies, VCR was found to lead to arrest in metaphase, not only of cells in or shortly prior to mitosis at the time of VCR administration, but also of the majority of cells which had at this time been in the S and G₂ phase.     

CELL KINETIC ALTERATIONS IN NORMAL AND NEOPLASTIC CELL POPULATIONS IN VITRO AND IN VIVO FOLLOWING VINCRISTINE*. A REPLY TO DR CAMPLEJOHN'S REVIEW ARTICLE†

It is shown that the lethal action of vincristine (VCR) is dose-dependent and may occur at interphase and mitosis. In general, the VCR dose used to destroy cells must be approximately ten times higher than that used to arrest cells in mitosis at metaphase. There is strong evidence that cells can survive metaphase arrest by a sublethal dose of VCR either completing cytokinesis normally after metabolism of the drug or becoming polyploid because of an impaired mitotic spindle apparatus. These cells are not doomed to die, at least in some cell systems. Furthermore, there is strong evidence in three animal tumour systems (transplantable and autochthonous tumours) that VCR is able to induce in vivo partial synchronization of proliferating tumour cells and/or recruitment of resting cells into the proliferating compartment. Failures to induce partial synchrony in cell populations by VCR may be attributed to resistance to VCR or cytolysis or slow proliferation of cells in badly vascularized tumours. Chemotherapy after synchronization seems to be effective as shown by non-randomized trials in bad-risk patients with solid tumours and acute leukaemias. In a randomized co-operative trial results of the two-drug synchronization protocol in patients with non-Hodgkin's lymphoma of high grade malignancy were statistically better than those of a four-drug protocol (COPP) established empirically. The two-drug protocol was equally effective as the four-drug protocol in Hodgkin's disease. Side-effects were less pronounced with the so-called synchronization scheme.     

Markedly decreased binding of vincristine to tubulin in vinca alkaloid-resistant Chinese hamster cells is associated with selective overexpression of alpha and beta tubulin isoforms

Vinca alkaloids are among a number of cytotoxic agents which target tumor cell microtubules. Studies described herein document the basis for one form of acquired resistance to these plant alkaloids involving an alteration of tubulin in a variant (DC-3F/VCRd-5L) of DC-3F Chinese hamster cells. Our results revealed a markedly decreased binding of [(3)H]vincristine (VCR) to tubulin extracted from this variant compared to tubulin extracted from wild-type DC-3F cells. This was quantitated as a 10- to 15-fold decrease in on-rate in the presence of GTP for the [(3)H]VCR associating with tubulin in cell-free cytosol and a 10-fold increase in off-rate for GTP-dependent dissociation of the [(3)H]VCR-tubulin complex. Quantitative RT-PCR and nucleotide sequencing of poly(A)(+) RNA also carried out with variant and wild-type DC-3F cells documented a different pattern of relative expression, but no base pair differences in the open reading frame of the three alpha and beta tubulin isoforms detected in each cell type. This was accounted for by selective overexpression of one alpha tubulin (alphaII) and two beta tubulin (betaI and betaIV) isoforms in the variant cells. These results would appear to provide an underlying basis for the large decrease in [(3)H]VCR binding by tubulin in these variant Chinese hamster cells and a major component of their acquired resistance to this vinca alkaloid.     

GTP influences the binding of vincristine in human tumor cytosols

The influence of GTP on the formation and stability of [3H] vincristine (VCR)-tubulin complexes in cytosols from two human rhabdomyosarcoma xenografts which have different sensitivities to VCR has been evaluated. After removal of endogenous GTP the initial rate of [3H]VCR binding and the maximal level of bound drug were 2- to 3-fold higher in the presence of 0.1 mM GTP than in its absence. Similarly, the stability of complexes was GTP-dependent. Complex formed from Rh18 tumors, only moderately sensitive to VCR, dissociated at 37 degrees in the absence of GTP with a half-time of 67 min; complex from Rh12 tumors (exquisitely sensitive to VCR) was more stable. Neither complex dissociated in the presence of 0.1 mM GTP over 2 hr examined.     

THE EFFECT OF VINCRISTINE ON MOUSE JEJUNAL CRYPT CELLS OF DIFFERING CELL AGE: DOUBLE LABELLING AUTORADIOGRAPHIC STUDIES USING 3H- AND 14C-TdR

The mechanism of action of the alkaloid vincristine (VCR) has been investigated in vitro on HeLa cells in culture and in vivo on jejunal crypt cells of the mouse. The in vitro experiments with HeLa cells show that VCR affects not only mitotic but also interphase cells. The VCR-affected cells first continue their passage through the cell cycle undisturbed but after reaching mitosis they are arrested in metaphase. This agrees well with the results obtained by Madoc-Jones & Mauro (1968) and Madoc-Jones (1973) on synchronized cell cultures. Until now there has been no investigation of the mechanism of action of VCR in vivo. This is due to the absence of a suitable technique for synchronization in vivo. The present study is based on a method which permits the assessment of the VCR sensitivity as a function of the cell age without synchronization in the usual sense. The jejunal crypt epithelium of the normal mouse was double labelled with ³H- and ¹⁴C-thymidine (TdR) in such a way as to produce a narrow subpopulation of crypt cells with a maximum age difference of 1 hr. On autoradiographs these cells can be distinguished by their characteristic labelling from other cells. As this ‘pseudo’-synchronized subpopulation passes through the cycle the effect of VCR can be studied, i.e. one can analyse the effect in well-defined time intervals of the cycle. The results show that the effect of VCR is the same in vivo as in vitro. The crypt cells which are affected by VCR in interphase continue their passage through the cycle, but upon entering mitosis they are arrested in metaphase. VCR has, at the concentration used in the present study, no effect on the duration of the S and G₂ phases. The necrotic cells seen after VCR application are formed from arrested metaphases.     

Combination of tunicamycin with anticancer drugs synergistically enhances their toxicity in multidrug-resistant human ovarian cystadenocarcinoma cells

Background  

The pharmacologic modulatory effects of the antibiotic, tunicamycin (TM), on multidrug-resistant human UWOV2 ovarian cancer cells are reported. The UWOV2 cell line was derived from a cystadenocarcinoma in a patient refractory to combination chemotherapy with actinomycin D, vincristine (VCR), cis-diaminedichloroplatinum (II) (CDDP) and doxorubicin (DXR). In an attempt to explain drug resistance in this cell line, we examined the effects of TM on their sensitivity to various anticancer drugs, the uptake, efflux and retention of [³H]VCR, and their ability to bind [¹⁴C]DXR and [³H]azidopine (AZD), a photoaffinity label of the multidrug transporter, P-glycoprotein (Pgp).     

Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells

The mechanism of reversal of resistance to Vinca alkaloids by cyclosporins is unclear. We investigated the molecular mechanism of reversal of Vinca alkaloid resistance by cyclosporin A (CsA) and its nonimmunosuppressive analog O-acetyl C9(1) CsA (SDZ 33-243) in multidrug resistant DC-3F/VCRd-5L Chinese hamster cells. CsA at 3 microM increased vincristine (VCR) sensitivity and almost totally reversed VCR resistance. SDZ 33-243 at 1 microM reduced the IC50 for VCR in resistant cells from 62.0 to 0.00062 microM. CsA and SDZ 33-243 at 10 microM increased [3H]vinblastine (VBL) accumulation in DC-3F/VCRd-5L cells by 27- and 22-fold, respectively. At 10 microM, these compounds also increased [3H]VCR accumulation by 3.5- and 4.0-fold, respectively. [3H]VCR uptake by membrane vesicles from DC-3F/VCRd-5L cells showed high and low affinity components with Michaelis-Menten kinetics, and apparent Km values were 0.140 +/- 0.0523 and 24.8 +/- 6.67 microM, respectively. Kinetic analysis of [3H]VCR uptake in membrane vesicles in the presence of 0.2 microM CsA revealed that CsA competitively inhibited the high affinity [3H]VCR uptake with an apparent inhibition constant (Ki) of 0.126 +/- 0.0173 microM. In addition, CsA and SDZ 33-243 inhibited VBL photoaffinity labeling of P-glycoprotein in a dose-dependent manner, with half-maximum inhibition at 0.5 and 0.4 microM, respectively, compared with that of VBL at 0.6 microM. These data confirm that cyclosporins modulate Vinca alkaloid resistance at least partially through interaction with P-glycoprotein.     

Overexpression of heme oxygenase-1 in microenvironment mediates vincristine resistance of B-cell acute lymphoblastic leukemia by promoting vascular endothelial growth factor secretion

Chemoresistance often causes treatment failure of B-cell acute lymphoblastic leukemia (B-ALL). However, the mechanism remains unclear at present. Herein, overexpression of heme oxygenase-1 (HO-1) was found in the bone marrow stromal cells (BMSCs) from B-ALL patients developing resistance to vincristine (VCR), a chemotherapeutic agent. Two B-ALL cell lines Super B15 and CCRF-SB were cocultured with BMSCs transfected with lentivirus to regulate the expression of HO-1. Silencing HO-1 expression in BMSCs increased the apoptotic rates of B-ALL cell lines induced by VCR, whereas upregulating HO-1 expression reduced the rate. Cell cycle can be arrested in the G2/M phase by VCR. In contrast, B-ALL cells were arrested in the G0/G1 phase due to HO-1 overexpression in BMSCs, which avoided damage from the G2/M phase. Vascular endothelial growth factor (VEGF) in BMSCs, as a key factor in the microenvironment-associated chemoresistance, was also positively coexpressed with HO-1. VEGF secretion was markedly increased in BMSCs with HO-1 upregulation but decreased in BMSCs with HO-1 silencing. B-ALL cell lines became resistant to VCR when cultured with VEGF recombinant protein, so VEGF secretion induced by HO-1 expression may promote the VCR resistance of B-ALL cells. As to the molecular mechanism, the PI3K/AKT pathway mediated regulation of VEGF by HO-1. In conclusion, this study clarifies a mechanism by which B-ALL is induced to resist VCR through HO-1 overexpression in BMSCs, and provides a novel strategy for overcoming VCR resistance in clinical practice.     

天然甾体皂甙化合物的抗肿瘤活性

采用MTT法,以长春新碱(VCR)为阳性对照,研究了6种从菝葜属植物中分离提取的天然甾体皂甙化合物对肝癌SMMC-7721、人宫颈癌HeLa和胃腺癌MGc80-3细胞生长的抑制作用.结果显示;6种甾体皂甙抗肿瘤活性与其化学结构密切相关,对三种癌细胞的抑瘤作用强度相同,抑癌活性的顺序为:薯蓣皂甙>VCR>SQD_4>SQD_3>M_1>SQD_1,甲基原薯蓣皂甙.甾体骨架的差异性是决定这类化合物抗肿瘤活性的主要因素.     

The effect of 5'-(N,N-dimethyl)-amiloride on cytotoxic activity of doxorubicin and vincristine in CEM cell lines

Intracellular pH (pHi) plays an important role in anticancer drug accumulation in cancer cells. Resistant cells often express membrane P-glycoprotein responsible for active drug extrusion and participating in increased pHi. In the present paper, we report on the influence of Na+/H+-exchanger inhibitor, 5'-(N,N-dimethyl)-amiloride (AMI), on the cytotoxic effects of doxorubicin (DOXO) and vincristine (VCR) in the parental CEM, and resistant CEM/DNR and CEM/VCR cell lines. The obtained results revealed a potentiating effect of AMI to both anticancer drugs in parental CEM line. However, AMI did not significantly potentiate the effect of DOXO or VCR in resistant CEM cell lines. We conclude, that inhibition of Na+/H+-exchanger by AMI is not sufficient for reversal of drug resistance in the tested CEM/DNR and CEM/VCR cell lines and the possible change in pHi does not affect the mechanisms of cell resistance.     

On down-regulation of the immune response to metastatic malignant melanoma

Treatment of metastatic malignant melanoma with interferon alpha (IFNalpha) results in objective remission in approximately 15% of patients. In a previous investigation, we found that about 50% of the patients achieved at least minor or short-lived remissions. In some tumours extensive areas of regressive tumour change occurred. However, even in these areas remnants of tumour cells were generally found. The short duration of the immune response in some patients and the incomplete eradication of the tumour can be due either to selection of non-immunogenic tumour cells or to down-regulation of the immune reactivity to the tumour. In the present paper, the expression of the zeta chain of the T cell receptor in CD3+ lymphocytes and the expression of CD28 in CD3+, CD4+ and CD8+ lymphocytes was studied in resectable melanoma metastases from 20 treated (IFNalpha or IFNalpha in combination with cisplatinum and dacarbazine) and 16 untreated patients. A double-staining technique was used, and the occurrence and distribution of lymphocytes showing down-regulation of the zeta chain or CD28 were separately registered in different areas of the metastases: close to the tumour cells in areas of unaffected tumour growth, in areas with regressive tumour changes, in areas with marked fibrosis and in stromal areas with densely packed lymphocytes. CD3+ zeta lymphocytes were found in all metastases, but their number and distribution varied considerably. Down-regulation of the zeta chain was most often found in areas of regressive changes. In contrast, T lymphocytes infiltrating close to the tumour cells had a stronger expression of the zeta chain (P = 0.016). Down-regulation was also found in stromal areas of densely packed lymphocytes and in areas of fibrosis. The pattern of down-regulation of CD28 in various subsets of lymphocytes was similar to that of zeta chain. The same pattern of down-regulation of CD28 and the zeta chain was found in both untreated and treated patients, indicating that the down-regulation is not due to treatment but to the release of immunosuppressor factors from areas with high tumour cell density or extensive destruction of tumour cells. These results concur well with the view that IFNalpha treatment can result in immune-mediated tumour cell destruction early in the treatment period and that this immune response to the tumour can be followed by immunosuppression within a few weeks.     

Wilms' tumour: adjuvant treatment with actinomycin D and vincristine.

In an unselected series of 49 children with Wilms'' tumour treated in 1969-74 the 5-year relapse-free survival and survival rates were 78% and 81%, respectively, whereas in the series of children treated in 1963-68 the corresponding rates were 49% and 70%. The significant improvement in the relapse-free survival rate was a result of adjuvant treatment with actinomycin D and vincristine (AMD + VCR), which, in some patients, eradicated occult metastatic disease. In the treatment of lung metastases the combination of whole-lung irradiation and maintained chemotherapy with AMD + VCR proved excessively toxic: in 5 of 11 patients acute diffuse pneumonitis developed, and it was fatal in 3. Adjuvant AMD + VCR therapy is advocated in all patients with Wilms'' tumour except children less than 12 months old with a tumour of moderate size, limited to the kidney and completely resectable.