Parameters affecting the ex vivo cytotoxic drug sensitivity of human hematopoietic cells

The drug sensitivity of normal cells provides a baseline for determining the therapeutic index, and therefore the effectiveness, of cytotoxic drugs, yet little is known about the factors that affect normal cell chemosensitivity. Some parameters are known to have a profound effect on tumor cell sensitivity. The purpose of this study was to determine how cytotoxic drug sensitivity of hematopoietic cells isolated from cancer patients was affected by various parameters. These included previous chemotherapy (yes or no), sex, age, tumor type (leukemias or solid tumors), sample source (blood, bone marrow, serous effusions, or tumor biopsies) and predominant cell lineage (lymphoid, myeloid, macrophage, or mixed). Mononuclear cells isolated from blood, bone marrow, serous effusions, and tumor biopsies were incubated for four days with a median of 16 drugs. The differential staining cytotoxicity assay, an ex vivo apoptotic drug sensitivity test in which cell survival is determined morphologically, was used to assess normal hematopoietic and tumor cell response to cytotoxic drugs. One hundred forty-six specimens yielded hematopoietic cell chemosensitivity results with 3-36 drugs. Compared with tumor cells, there was far less interpatient variation in chemosensitivity of hematopoietic cells. Mean hematopoietic cell drug sensitivity showed little variation due to previous chemotherapy, sex, age, tumor type, and sample source or cell lineage. We therefore concluded that cytotoxic drug sensitivity of hematopoietic cells from a variety of sources could be used for assessment of therapeutic index. Drug therapeutic index results are a valuable tool in identifying novel cytotoxic agents and individually tailored chemotherapy regimens.     

Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells

Fibroblast growth factor (FGF) belongs to a family of heparin-binding polypeptides and shows multiple functions including cell proliferation, differentiation, survival and motility. The expression of FGF receptors is widely distributed on different hematopoietic progenitor cells and stromal cells, and FGFs play an important role in hematopoietic stem cell homeostasis. FGFs have been shown to sustain the proliferation of hematopoietic progenitor cells, maintaining their primitive phenotype. Basic FGF (bFGF, FGF-2) stimulates the formation of an adherent stromal cell layer in human long-term bone marrow cultures, and promotes hematopoietic cell development. FGF-2 has also been shown to synergize with other hematopoietic growth factors to enhance in vitro colony formation by several classes of hematopoietic progenitor cells. Results of ex vivo expansion and clinical trials to date suggest that hematopoietic cells cultured under stroma-free cytokine combination conditions may be insufficient to restore hematopoiesis after a myeloablative conditioning regimen, although some recent trials demonstrated an improvement in engraftment and a reduction of the period of pancytopenia, especially neutrophils and platelets, after transplantation. A recent study by our group demonstrated that FGF-2 is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion. These observations could be useful in reducing the long period of severe thrombocytopenia that occurs frequently after umbilical/placental cord blood transplantation. The development of more effective amplifying systems for hematopoietic stem/progenitor cells can be expected since FGFs have multiple functions in hematopoiesis.     

Restoration of sensitivity to oxazaphosphorines by inhibitors of aldehyde dehydrogenase activity in cultured oxazaphosphorine-resistant L1210 and cross-linking agent-resistant P388 cell lines

The sensitivity of cultured L1210 and P388 cells sensitive (L1210/0, P388/0) and resistant (L1210/OAP, P388/CLA) to oxazaphosphorines, to 4-hydroperoxycyclophosphamide, ASTA Z-7557, phosphoramide mustard, and acrolein was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. The L1210/OAP cell line is resistant specifically to the oxazaphosphorines; P388/CLA cells are partially cross-resistant to other cross-linking agents. All four inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines, 4-hydroperoxycyclophosphamide and ASTA Z-7557, against L1210/OAP and P388/CLA cells; in the presence of a sufficient amount of inhibitor, sensitivity was essentially fully restored in both cases. The inhibitors did not potentiate the cytotoxic action of the nonoxazaphosphorines, phosphoramide mustard and acrolein, against these cell lines. The cytotoxic action of the oxazaphosphorines and nonoxazaphosphorines against L1210/0 and P388/0 cells was not potentiated by any of the aldehyde dehydrogenase inhibitors. Inhibitors of xanthine oxidase or aldehyde oxidase activities did not potentiate the cytotoxic action of the oxazaphosphorines against L1210/OAP cells. These observations strongly suggest that (a) aldehyde dehydrogenase activity is an important determinant with regard to the sensitivity of a cell population to the oxazaphosphorines; (b) L1210/0 and P388/0 cells lack the relevant aldehyde dehydrogenase activity; (c) the phenotypic basis for the resistance to oxazaphosphorines by L1210/OAP cells is aldehyde dehydrogenase activity; and (d) the major reason that P388/CLA cells are resistant to oxazaphosphorines is aldehyde dehydrogenase activity.     

Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells.

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5'-6'-dihydrofluorouracil, which is further metabolized to fluoro-beta-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG-DPD-IRES-Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU-resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10(-6) M) for 14 days also resulted in an increased number of 5-FU-resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity.     

The AFT024 stromal cell line supports long-term ex vivo maintenance of engrafting multipotent human hematopoietic progenitors.

The immortalized murine stromal cell line AFT024 has been reported to maintain human hematopoietic progenitors in an undifferentiated state in vitro. In the current studies the beige/nude/xid (bnx) mouse in vivo xenograft model was used to examine the engraftment and multilineage generative potential of human hematopoietic progenitors after 2-3 weeks growth on AFT024 stroma, in comparison to primary stromal monolayers derived from post-natal human bone marrow. Eight to 12 months after transplantation of human CD34+CD38- cells from umbilical cord blood, cultured on AFT024 vs human stroma for 2-3 weeks, the murine bone marrow was harvested and analyzed for the presence of human myeloid and lymphoid cells. The mean percent engraftment of total human hematopoietic cells in the murine marrow was significantly higher after co-cultivation on AFT024 than on human stroma. Human myeloid and lymphoid lineage cells were detected in all mice. However, engraftment of myeloid lineage cells (CD33+), B lymphoid (CD19+), and T lymphoid cells (CD4+and CD8+) were significantly higher after co-cultivation of the human cells on AFT024 than on human stroma, prior to transplantation. Interestingly, the length of time in culture did not significantly affect the engraftment of the myeloid and T lymphoid lineage progenitors, but the percentage of B lymphoid lineage engraftment decreased significantly between 2 and 3 weeks of co-cultivation on both types of stroma. Cells with a primitive phenotype (CD45+/CD34-/CD38- and CD45+/CD34-/lin-) and cells with the capacity to generate secondary human CFU after recovery from the bnx bone marrow were maintained at significantly higher levels during culture on AFT024 stroma than on human stroma. The current studies demonstrate that the AFT024 murine stromal cell line supports the ex vivo survival and maintenance of human hematopoietic progenitors that are capable of long-term multilineage reconstitution for 2-3 weeks ex vivo, to levels superior to those that can be obtained using human stromal cells.     

Variable effects of tamoxifen on human hematopoietic progenitor cell growth and sensitivity to doxorubicin

To determine the influence of tamoxifen on the drug sensitivity of normal human hematopoietic progenitor cells, T-cell- and adherent-cell depleted human bone marrow mononuclear cells (T^–, Ad^–) were exposed in vitro to 5 M tamoxifen for 24 h. The effects of tamoxifen were highly variable, as exposure to tamoxifen produced an increase (97%±12.3%) in the growth of day-12 committed myeloid progenitors (CFU-GM) in only four of ten experiments utilizing bone marrow from different donors. When T^–, Ad^– myeloid progenitor cells treated with tamoxifen were subsequently exposed to doxorubicin, 7 of 14 experimental samples studied demonstrated a net increase in the number of surviving clonogenic cells as compared with cells exposed to doxorubicin alone. Tamoxifen also stimulated the growth of a more purified (CD34⁺-selected) progenitor cell population in four of four experiments (by 62.5%±4.9%) but did not increase the survival of these cells upon exposure to doxorubicin; in fact, in five of ten experimental samples, tamoxifen enhanced cell sensitivity to doxorubicin. Taken together, these observations indicate that tamoxifen produces variable stimulation of committed myeloid progenitor cell growth in vitro. Furthermore, while under some circumstances, tamoxifen appears to have the capacity to enhance CFU-GM survival in the presence of doxorubicin, this drug combination may also result in enhanced toxicity to normal bone marrow progenitors.Abbreviations CFU-GM granulocyte-macrophage colony-forming units - T^–, Ad^– T-cell- and adherent-cell-depleted bone marrow mono-nuclear cells     

Effect of lymphokine-activated killer cell fraction on the development of human hematopoietic progenitor cells

Lymphokine-activated killer (LAK) cells from cultures of human peripheral blood mononuclear cells with recombinant interleukin-2 (rIL-2) have been clinically used in adoptive immunotherapy for cancer patients. To study their influence on human hematopoiesis, the LAK cell fraction was cocultured with marrow nonphagocytic cells from normal subjects in an assay system of hematopoietic progenitors. The fraction suppressed colony growth from relatively mature erythroid progenitors in a dose-dependent manner. Although unactivated cells, which were produced without IL-2, augmented the growth of early erythroid progenitors, the LAK cell fraction did not. This fraction suppressed colony growth from mature granulocyte-macrophage progenitors (day 7 CFU-GM) especially with an 18-h preincubation prior to coculture. It also suppressed both immature granulocyte-macrophage progenitors (day 14 CFU-GM) and multipotential hematopoietic progenitors. The suppressive effects were observed on colony growth from autologous marrow cells as well as allogeneic marrow cells. The suppression of day 7 CFU-GM colony growth by supernatants due to preincubation with marrow cells and the LAK cell fraction suggested that the humoral factor contributes to the suppression by the LAK cell fraction. These data suggest that the LAK cell fraction suppresses the development of human hematopoietic progenitor cells.     

Role of aldehyde dehydrogenase in the protection of hematopoietic progenitor cells from 4-hydroperoxycyclophosphamide by interleukin 1 beta and tumor necrosis factor.

Preincubation of human bone marrow cells with interleukin 1 beta (IL-1) and tumor necrosis factor alpha (TNF-alpha) for 20 h can protect early progenitor cells from 4-hydroperoxycyclophosphamide (4-HC) toxicity. In this report, we have studied the mechanism for such protection. We examined the effect of the length of incubation time and found that preincubation for at least 20 h with IL-1 and TNF-alpha is needed for significant protection. The addition of 2 micrograms/ml cycloheximide, a protein synthesis inhibitor, during the 20-h preincubation completely abolished the protection observed for all colony-forming cells. In order to study the role of aldehyde dehydrogenase (ALDH), an enzyme which inactivates 4-HC, we used diethylaminobenzaldehyde, an inhibitor of ALDH. Diethylaminobenzaldehyde was added during the last 10 min of the 20-h preincubation with IL-1 and TNF-alpha. Diethylaminobenzaldehyde prevented the protection of colony-forming cells from 4-HC. Finally, using the same protection assay system, we showed that a 20-h preincubation with IL-1 and TNF-alpha can also protect early progenitor cells from phenylketophosphamide, an analogue of 4-HC which is resistant to inactivation by ALDH. From these studies, we conclude that preincubation with IL-1 and TNF-alpha for at lest 20 h is required for the protection of early progenitor cells from 4-HC. During that time period, protein synthesis, specifically aldehyde dehydrogenase synthesis, is critical for the protection from 4-HC. Preincubation with IL-1 and TNF-alpha also protects early progenitors from phenylketophosphamide. Because phenylketophosphamide cannot be metabolized by ALDH, the reason for this protection must be due to other, as yet unidentified, mechanisms.     

血液肿瘤细胞对氧化砷的敏感性与其抗氧化能力的关系

目的　探讨血液肿瘤细胞对三氧化二砷 (As2 O3 )的敏感性和细胞抗氧化能力的关系。方法　应用 9个血液肿瘤细胞系 ,通过细胞活力、形态学和流式细胞仪检测细胞凋亡 ,并测定细胞系的谷胱甘肽 (GSH)含量和 4种抗氧化酶的活性。结果　除了HL 6 0、U937、K5 6 2和Jurkat细胞外 ,其他5个细胞对As2 O3 诱导的凋亡敏感。与敏感细胞系比较 ,As2 O3 耐受细胞系存在较高的GSH含量和(或 )过氧化氢酶活性。谷胱甘肽过氧化物酶、谷胱甘肽S转移酶和超氧化物歧化酶活性与细胞对As2 O3 诱导凋亡效应敏感性无明显相关。结论　细胞内GSH水平和过氧化氢酶的活性是决定血液肿瘤细胞对As2 O3 敏感性的重要因素。     

The rho-kinase inhibitors Y-27632 and fasudil act synergistically with imatinib to inhibit the expansion of ex vivo CD34(+) CML progenitor cells.

Evidence from cell line-based studies indicates that rho-kinase may play a role in the leukaemic transformation of human cells mediated by the BCR/ABL tyrosine kinase, manifest clinically as chronic myeloid leukaemia (CML). We therefore employed two separate inhibitors, Y-27632 and fasudil, to inhibit the activity of rho-kinase against ex vivo CD34(+) cells collected from patients with CML. We compared the effects of rho-kinase inhibition in those cells with the effects of direct inhibition of BCR/ABL using the specific inhibitor imatinib. We found that inhibition of rho-kinase inhibited the effective proliferation, and reduced survival of CML progenitor cells. When combined with imatinib, rho-kinase inhibition added to the anti-proliferative and pro-apoptotic effects of the BCR/ABL inhibitor. Our studies may indicate therapeutic benefit in some cases for the combination of rho-kinase inhibitors with imatinib.     

Effect of imatinib mesylate on chronic myelogenous leukemia hematopoietic progenitor cells

Treatment of chronic myelogenous leukemia (CML) has been greatly enhanced by the development of Imatinib mesylate, a specific inhibitor of the BCR/ABL tyrosine kinase. While it is clear that imatinib effectively targets BCR/ABL positive hematopoietic cells, studies examining its effect on primitive hematopoietic progenitors are much more limited. As CML arises in a primitive hematopoietic progenitor cell, it is especially important to understand the effect of imatinib on these cells. Here we review studies investigating the effect of imatinib on the proliferation and viability of primitive and committed hematopoietic progenitors in CML. We describe evidence that BCR/ABL positive progenitors may persist in patients responding to imatinib and discuss problems of resistance to imatinib. Finally we discuss studies evaluating new approaches to overcome resistance of CML progenitor cells to imatinib.     

IL-6/sIL-6R对人脐血CD34+细胞体外扩增的作用

背景和目的:造血干细胞具有自我更新、多向分化与重建长期造血的潜能,因此,造血干细胞广泛应用于干细胞移植、免疫治疗、基因治疗等领域.胎儿脐带血中富含造血干细胞,但单份脐带血中造血干细胞的数量有限,不能充分满足临床和科研需要,因此在体外培养使脐带血干/祖细胞数量扩增的研究日益受到重视.已知一些细胞因子可以在体外培养中使脐血干/祖细胞大量扩增.新近发现IL-6/sIL-6R(可溶性IL-6受体)或其融合蛋白可促使脐带血CD34+细胞中CD34+gp130+IL-6R-细胞亚群在体外培养中大量扩增.本实验旨在观察IL-6/sIL-6R在脐带血CD34+细胞体外扩增中的作用,并探讨合适细胞因子组合.方法:脐血CD34+细胞用Mini MACS分离,然后在含有不同细胞因子组合的液体培养基中体外培养7天或14天,培养前后分别进行有核细胞计数、用FCM(流式细胞术)测定CD34+细胞比例计算CD34+细胞总数及进行CFU-GM集落培养.根据不同细胞因子组合分为空白对照组,SCF组,SCF+IL-6/sIL-6R组,SCF+FL+IL-6/sIL-6R组,SCF+FL组.结果:空白对照组和SCF组CD34+细胞数量在培养7天或14天后明显下降.SCF+IL-6/sIL-6R组培养7、14天分别使有核细胞及CD34+细胞绝对数扩增(7.1±2.4)倍、(39.0±14.0)倍及(1.8±0.7)倍、(4.8±2.4)倍;SCF+FL+IL-6/sIL-6R组(16.5±5.7)倍、(110.0±28.0)倍及(3.5±1.5)倍、(10.2±4.2)倍;SCF+FL组(17.3±3.8)倍、(104.0±21.0)倍及(3.6±2.1)倍、(8.4±3.5)倍.上述三组与对照组及SCF组差异均有显著性(P＜0.01),其中SCF+FL+IL-6/sIL-6R组和SCF+FL组扩增效果优于SCF+IL-6/sIL-6R组(P＜0.01),但是SCF+FL+IL-6/sIL-6R组和SCF+FL组之间差异无显著性(P＞0.05).增加sIL-6R浓度,当sIL-6R浓度为400 ng/ml时,细胞培养7天,SCF+FL+IL-6/sIL-6R组分别使有核细胞及CD34+细胞绝对数扩增(24.0±4.8)倍、(5.6±1.2)倍,优于SCF+FL组(P＜0.05).结论:IL-6/sIL-6R可以和SCF、FL产生协同作用,使人脐血CD34+细胞在体外扩增、并维持其分化潜能.但这种协同作用对sIL-6R的浓度存在一定依赖性.     

Mobilization of hematopoietic stem and progenitor cells using inhibitors of CXCR4 and VLA-4

Successful hematopoietic stem cell transplant requires the infusion of a sufficient number of hematopoietic stem/progenitor cells (HSPCs) that are capable of homing to the bone marrow cavity and regenerating durable trilineage hematopoiesis in a timely manner. Stem cells harvested from peripheral blood are the most commonly used graft source in HSCT. Although granulocyte colony-stimulating factor (G-CSF) is the most frequently used agent for stem cell mobilization, the use of G-CSF alone results in suboptimal stem cell yields in a significant proportion of patients. Both the chemokine receptor CXCR4 and the integrin α(4)β(1) (very late antigen 4 (VLA-4)) have important roles in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid and reversible mobilization of hematopoietic stem cells into the peripheral circulation and is synergistic when combined with G-CSF. In this review, we discuss the development of small-molecule CXCR4 and VLA-4 inhibitors and how they may improve the utility and convenience of peripheral blood stem cell transplantation.     

Protective effects of human interleukin-1 on hematopoietic progenitor cells from L-phenylalanine mustard

The effects of interleukin-1 (IL-1) on protecting both human and murine bone marrow cells were studied using in vitro clonogenic assays, long-term bone marrow cultures and in vivo mouse studies. Incubation with 100 ng/ml human recombinant IL-1 beta for 20 hours prior to a one hour exposure to L-phenylalanine mustard (L-PAM) provided significant protection of bone marrow colony forming cells when compared to bone marrow cells not exposed to IL-1. Complete inhibition of colony formation was observed above 40 mu M L-PAM in the absence of IL-1 preincubation; whereas, colonies were still detectable in cultures which were initiated with IL-1-treated bone marrow cells. Similar results demonstrating greater protection with IL-1 incubation from L-PAM were seen when murine bone marrow cells were assayed for long-term culture-initiating cells. Furthermore, IL-1 protects long-term repopulating hematopoietic stem cells from L-PAM when studied using an in vivo irradiated mouse assay. In contrast, incubation with IL-1 does not protect colony formation by K562, KG-1 or HL-60 leukemic cell lines implying that protection by IL-1 may be selective. Finally, the protection observed by IL-1 preincubation could be abrogated by incubation with 50 mu M L-buthionine sulfoximine (BSO). This result indicates that IL-1 may increase the amount of glutathione in hematopoietic cells and be responsible for the observed protection from L-PAM.     

BCR-ABL alters the proliferation and differentiation response of multipotent hematopoietic cells to stem cell factor

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.     

Effects of L-phenylalanine mustard and L-buthionine sulfoximine on murine and human hematopoietic progenitor cells in vitro

The effects of L-buthionine sulfoximine (L-BSO) and L-phenylalanine mustard (L-PAM), alone and in combination, on human and murine marrow were explored using in vitro clonogenic assays to establish whether enhanced myelotoxicity might limit the clinical utility of this potent chemotherapeutic combination. One-h exposure to L-PAM produced significant concentration-dependent colony inhibition, with 70% inhibitory concentration (IC70) values ranging from 4.5 to 7.2 microM for all hematopoietic progenitors assayed. The combination of L-PAM plus 4500 microM L-BSO for 1 h did not effectively alter the IC70 values derived for L-PAM alone. In studies where marrow cells were pretreated with L-BSO for 4 h and then L-PAM for 1 additional h, the IC70 values were decreased in both murine and human marrow progenitors compared to the L-PAM control, suggesting modest potentiation of myelotoxicity. The potentiation is not so significant as to preclude human studies with this combination. One- to 5-h exposure of marrow cells from both species to 4500 microM L-BSO was only mildly myelotoxic, producing colony reductions of 22-49%. However, continuous exposure to L-BSO produced concentration-dependent colony inhibition, with IC70 values of 70, 84, and 43 microM for murine colony-forming units-granulocyte/macrophage, blast-forming units-erythroid, and colony-forming unit-erythroid, respectively.     

The mechanism of differential sensitivity to methotrexate of normal and malignant human epidermal cells

Summary Squamous carcinoma cells are much more sensitive (>10⁴ times) to the cytotoxic effects of methotrexate (MTX) and 5-fluorodeoxyuridine (FUDR) than are normal human keratinocytes as measured by cell growth. Among the drugs tested, this phenomenon was found to be specific for MTX and FUDR, since arabinosylcytidine (ARA-C), 13-bis-chloroethylnitrosourea (BCNU), and daunomycin failed to show differences in inhibition between the normal and malignant cell lines. Drug uptake studies did not reveal a significant difference in MTX intracellular levels between malignant and normal epidermal cell lines at 60 min. Thymidine (TdR) salvage was assessed by examining the effects of the presence of 3 M TdR on MTX-induced cytotoxicity. On the withdrawal of TdR, normal cells demonstrated an increased level of inhibition amounting to 4 orders of magnitude, whereas the squamous-cell carcinoma cells showed no change in sensitivity. Interestingly, the immortal nontumorigenic keratinocyte line (NM-110) was similarly not rescued by the addition of TdR. The high degree of sensitivity to MTX shown by squamous-cell carcinoma (SCC) and NM-110 cells is attributable to a significant diminution of their ability to use exogenous TdR as compared with that of normal keratinocytes and might be indicative of a biochemical change associated with cellular immortality.This work was supported in part by grants from the Josephine M. Lilly Memorial Melanoma Research Fund, the National Foundation for Cancer Research, and the National Institutes of Health (CA 24988). This work was presented in part at The Society for Investigative Dermatology Tricontinental Meeting, April 1989, Washington, D. C.