Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.     

Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haemopoietic cells in NOD/SCID mice transplanted with human cord blood

Time course studies of sublethally irradiated non-obese mice with severe combined immunodeficiency (NOD/ SCID mice) transplanted intravenously with 10(7) human cord blood cells showed a rapid and parallel regeneration of human erythroid, granulopoietic, megakaryopoietic and B-lymphoid progenitors, as well as more primitive subpopulations of CD34+ cells (defined by their multi-lineage in vitro colony-forming ability, coexpression of Thy-1, or functional activity in long-term culture-initiating cell [LTC-IC] assays), in the marrow, spleen and blood. Maximum numbers of human cells were reached within 6 weeks and were then sustained for another 18-20 weeks. 3H-thymidine suicide studies showed all types of in vitro clonogenic human progenitors tested and the human LTC-IC to be proliferating in vitro throughout this period. A 2-week course of injections of human Steel factor, interleukin-3, granulocyte-macrophage colony-stimulating factor and erythropoietin given just prior to assessment of the mice had no effect on any of these human engraftment parameters. 4-6 weeks post-transplant, the marrow of primary NOD/SCID recipients contained human cells that were able to regenerate lymphopoiesis and/or myelopoiesis in secondary irradiated NOD/SCID mice. These findings establish a baseline for the kinetics of engraftment, multi-lineage differentiation and self-renewal of human cord blood stem cells in this xenogeneic transplant model and thus set the stage for future studies of their regulation in vivo.     

Evidence for a 40-Hz oscillatory short-term visual memory revealed by human reaction-time measurements.

Four experiments show that presentation of a synchronous premask frame within a 40-Hz, flickering premask matrix primes subsequent detection of a Kanizsa-type square by generation of a 40-Hz prime. Reaction time (RT) priming effects indicated a 150–200-ms prime duration following premask display. RTs were also found to be sensitive to the phase relationship between offset of the premask display relative to the onset time of the target: Priming effects were maximal when the target was presented out of phase with premask presentation (i.e., at interstimulus intervals displaced by 180° relative to the 40-Hz rhythm of premask-frame presentation). Taken together, these results demonstrate the existence of a very short-term visual memory that oscillates at 40 Hz. The findings are discussed in the context of complementary psychological and neurophysiological findings related to visual-object coding and the role of gamma-band activity in the brain. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence that ferritin is UV inducible in human skin: part of a putative defense mechanism

The low-affinity penicillin-binding protein (PBP) 5 is the main beta-lactam target and is responsible for resistance to this class of antibiotics in Enterococcus faecium. The PBP 5 variants of 15 clinical isolates (including 8 resistant to vancomycin) with different levels of beta-lactam resistance were analyzed. Most of the highly beta-lactam-resistant isolates produced small quantities of PBP 5 of low affinity. This was associated with particular amino acid substitutions: an Ala or Ile for Thr-499, a Glu for Val-629, and a Pro for Ser-667. A change of Met-485 to Thr or Ala (adjacent to the conserved SDN box) was observed in isolates with MICs of ampicillin of 64 or 128 microg/mL, respectively. In the 2 most resistant isolates, with MICs of ampicillin of 256 microg/mL, an additional Ser was present just after Ser-466. Thus, particular point mutations in PBP 5 and combinations thereof may lead to high-level beta-lactam resistance in E. faecium.     

Perforin-deficient CD8+ T cells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-gamma but requires TNF-alpha

CD8+ T cells are effective mediators of immunity against Listeria monocytogenes, but the mechanisms by which they provide antilisterial immunity are poorly understood. CD8+ T cells efficiently lyse target cells in vitro by at least two independent pathways. To test the hypothesis that CD8+ T cell-mediated immunity to L. monocytogenes is dependent on perforin or CD95 (Fas, Apo-1), we used C57BI/6 (B6) and perforin-deficient (PO) mice to generate CD8+ T cell lines specific for the L. mono cytogenes-encoded Ag listeriolysin O (LLO). Both lines specifically produce IFN-gamma and TNF-alpha, and mediate target cell lysis in vitro. Cytolysis mediated by the PO-derived CD8+ T cell line is delayed relative to the B6-derived line and is completely inhibited by anti-CD95 Abs. In vivo, PO-derived CD8+ T cells provide specific antilisterial immunity in B6 hosts, CD95-deficient hosts, and IFN-gamma-depleted hosts. However, PO-derived CD8+ T cells fail to provide antilisterial immunity in hosts depleted of TNF-alpha. These results indicate that single Ag-specific CD8+ T cells derived from PO mice can mediate antilisterial immunity by a mechanism that is independent of CD95 or IFN-gamma, but requires TNF-alpha.     

Evidence for synthesis and release of catecholamines by human amniotic epithelial cells

The present study investigated the presence, possible synthesis and release of catecholamines (CA) by human amniotic epithelial cells (HAEC) using HPLC with electrochemical detection. The presence of CA was indicated by the detection of norepinephrine (NE), dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in extracts of cultured HAEC. Incubation of HAE cells in medium supplemented with 1-tyrosine (CA precursor) and tetrahydrobiopterin (tyrosine hydroxylase cofactor) significantly increased the production of catecholamines, suggesting CA synthesis by HAEC. In contrast, pharmacological inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (MPT) significantly reduced CA production, further confirming CA synthesis by HAEC. Catecholamines were also detected in the cell incubation media, demonstrating the ability of HAEC to spontaneously secrete CA. Moreover, incubation of cells with 50 mM K+ for 10 min increased the amount of CA released into the medium. Additionally, the detection of DOPAC, a primary metabolite of DA, in HAEC strongly indicates that these cells contain DA metabolizing enzymes. The present results suggest that HAEC synthesize and release CA. These cells may be a possible candidate for transplantation therapy of neurodegenerative diseases such as Parkinson's disease and also may serve as a model to study the aspects of catecholaminergic activity.     

B-cell activation in vitro by helper T cells specific to a protein region that is recognized both by T cells and by antibodies

The effects of Trypanosoma evansi on efferent lymphocyte phenotypes draining from a lymph node primed with Pasteurella haemolytica vaccine were studied in sheep. The prefemoral efferent lymphatic ducts of the infected sheep along with those of two uninfected sheep were surgically cannulated. Lymph was collected and lymphocytes recovered from it analysed by two-colour indirect immunofluorescence staining and cytofluoremetry in a fluorescence activated cell analyser (FACSCAN). The study showed the appearance and persistence of T. evansi in the efferent lymph for a long period of time and the appearance of CD4+CD8+ (double positive, DP) T lymphocytes in the efferent lymph of infected animals. The infection also resulted in increases in CD5+ B cells in the prefemoral efferent lymph. In addition, there were decreases in the output of conventional B cells, CD5+ and CD4+ T cell subsets but large increases in CD8+ cells followed by terminal depletion of all cell subsets. In contrast, inoculation of sheep with pasteurella vaccine antigen alone produced little alterations in the proportions, but large increases in the numbers of all T cell subsets except that of CD8+ cells which also showed little variation; and there was a concurrent increase in the numbers and proportions of efferent B cells. In addition, the abnormal expression of DP and CD5+ B cells did not occur in the uninfected vaccinated sheep. It is concluded that these abnormal changes in the kinetics of efferent lymphocyte phenotypes are likely to play a role in the genesis of the generalized immunosuppression seen in trypanosome-infected hosts.     

Characteristics of chromosomes in polarized normal human bronchial cells provide a blueprint for nuclear organization

In normal human terminally differentiated and polarized bronchial cells, the fluorescent "painting" technique (FISH) for all chromosomes (except Y) documented that each homologue of each chromosome occupies a distinct, separate domain within the nucleus. The homologues are distributed along the nuclear membrane. In most cells and chromosomes studied, the two homologues were not identical: one was usually more "compact" than the other which was more "open," displaying fiber-shaped extensions. The differences between the territories of homologues 1 and 7 were shown to be statistically valid (P < 0.0001 by Wilcoxon sign rank test), as has been previously documented for the two X chromosomes (Eils et al., 1996). In some parallel arrays of bronchial cells, the position of the chromosomes in the nuclei was either identical or formed a mirror image, suggesting that the position of the chromosomes in polarized nuclei may be constant. To confirm this observation, the angles formed by the two homologues in the polarized oval nuclei were measured for chromosomes 1, X, and 7. The measurements disclosed that, in about two-thirds of the nuclei, the two homologues formed angles of 150 degrees, 157 degrees and 148 degrees, nearly identical to those formed by the same three chromosomes in prometaphase rosettes of cultured diploid human fibroblasts (Nagele et al., 1995). In about one third of the nuclei, the same homologues formed angles of 89 degrees, 72 degrees, and 94 degrees, and occasionally an angle of 180 degrees. A three-dimensional computer reconstruction of the nuclei was performed using the data for the X chromosomes. By cinematographic technique, it could be documented that the angles separating the two homologues depended on the rotation of the nucleus along the axes X, Y, and Z. The cause of the rotation is speculative at this time. Because of the concordance of these data in terminally differentiated epithelial cells with prior observations on prometaphases of human diploid fibroblasts, it is suggested that the position of chromosomes in all human cells is constant throughout the cell cycle. The possible significance of these observations is discussed.     

Desensitization of hepatoma cells to insulin action. Evidence for a post-receptor mechanism

We have previously reported that incubation of rat hepatoma cells with insulin causes a complete and reversible loss of responsiveness to insulin. In order to determine the role of the insulin receptor in desensitization, we have examined the effect of insulin on insulin binding. Exposure of rat hepatoma cells to insulin causes a time-dependent decrease in insulin binding capacity which is detectable at 30 min and maximal at 4-6 h, after which time insulin binding remains 40-50% that of untreated cells. Scatchard analysis indicates that insulin causes a decrease in the number of receptors with little change in the binding affinity. Insulin-induced down regulation of receptors, observable at insulin concentrations as low as 3 ng/ml, is half-maximal at 10-20 ng/ml and is maximal at 100 ng of insulin/ml. When insulin is removed from the culture medium, the cells slowly recover insulin binding capacity; recovery is minimal at 2-4 h but nearly complete after 24 h. Recovery of insulin responsiveness, in contrast, is complete as early as 2 h after insulin is removed. The extent of down regulation of receptors (50-60%) is not sufficient to account for the complete insulin desensitization. In addition, recovery of maximal responsiveness to insulin occurs long before recovery of insulin binding. Therefore, insulin-induced desensitization to insulin is not caused by down regulation of receptors but must involve a post-receptor mechanism.     

Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable, long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation.     

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.     

Evidence that activation of human T cells by porcine endothelium involves direct recognition of porcine SLA and costimulation by porcine ligands for LFA-1 and CD2

In this study we present a comprehensive evaluation of the molecular interactions between human T cells and porcine aortic endothelial cells (PAEC) that contribute to human T cell activation. Binding assays demonstrated that porcine erythrocytes (E) and PAEC express ligand(s) for the human T cell glycoprotein CD2. Prior incubation of human T cells with a blocking monoclonal antibody directed against CD2 (alpha CD2-BL) completely inhibited T cell/E and T cell/PAEC interaction. Xenogeneic mixed lymphocyte reactions (XMLR) revealed that human PBMC, or highly purified T cells were activated by PAEC in the absence of human antigen-presenting cells (APC). Addition of alpha CD2-BL or alpha LFA-1 to these assays inhibited PAEC-mediated human T cell activation. Furthermore, we demonstrated that highly purified human CD4+ and CD8+ T cells proliferated in response to PAEC and that this response was blocked by monoclonal antibodies directed against LFA-1 and CD2. Addition of alpha SLA class I blocked the proliferation of CD8+ but not CD4+ T cells, indicating direct presentation of SLA class I antigens to human T cells. We have recently shown that expression of the human complement inhibitor (CD59) on PAEC (PAEC-LXSNCD59) rendered these cells resistant to human complement-mediated activation and lysis, suggesting that human CD59 expression on PAEC could be an effective therapy for hyperacute rejection (HAR). However, recent studies have shown that in addition to its role as a complement inhibitor, CD59 binds human T cell CD2 and contributes to T cell activation. We therefore examined whether human CD59 expression on PAEC augmented the human antiporcine T cell response. We demonstrated that human T cells do not display increased binding to or activation by PAEC-LXSNCD59 relative to PAEC controls. Taken together, our data establish that PAEC directly stimulate human T cells in vitro and that interactions between the human accessory molecules CD2, LFA-1 and their PAEC surface ligands contribute to human T cell activation. In addition, the expression of human CD59 on porcine donor organs may confer resistance to human complement-mediated HAR without exacerbating the human antiporcine cellular response.     

Exogenously provided peptides of a self-antigen can be processed into forms that are recognized by self-T cells

Major histocompatibility complex (MHC) class II molecules can present peptides derived from two different sources. The predominant source of peptide in uninfected antigen presenting cells (APCs) is from self-proteins that are synthesized within the cell and traffic through the MHC class II compartment. The other source of antigen is endocytosed proteins, which includes both self- and foreign proteins. Foreign protein antigens generate adaptive immune responses, whereas self-peptides stabilize the MHC class II heterodimer on the cell surface, allowing positive and negative selection of thymocytes. Therefore, self-antigens play an important normal role in shaping the T cell receptor repertoire as well as a pathological role in autoimmunity. To determine whether processing and presentation of self-antigens by MHC class II molecules differs depending on whether the antigen is supplied through synthesis within the cell or by endocytosis, we used a T cell clone against an Ealpha peptide presented by I-Ab to show that processing through these two routes can differ. We also show that mice can be tolerant to the epitope formed through the endogenous route, but responsive to the epitope that can be formed through endocytosis. This suggests that negative selection occurs primarily against antigens that are synthesized within the APC, and that endocytosed self-antigens could serve as autoantigens. Finally, we also demonstrate that lipopolysaccharide-activated B cells are defective for uptake, processing, and presentation of this self-antigen, and that this correlates with the increased expression of the costimulatory molecules B7.1 and B7.2. This may provide a model for studying the onset of an autoimmune response.     

Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions

Twenty-eight proximal phalangeal fractures secondary to low-velocity gunshot wounds in 27 patients treated by stable fixation were retrospectively reviewed. Definitive fixation was performed within 1 week of injury. Fractures were stabilized with either a plate, intramedullary spacer, or a combination of both. When necessary, supplemental fixation was achieved with cerclage wires or interfragmentary screws. Twenty fractures with bone loss or comminution were primarily supplemented with iliac crest bone graft. After surgery, the fingers were splinted in 90 degrees of metacarpophalangeal (MP) flexion. An aggressive supervised therapy program was initiated within 24 hours of surgery. The average length of follow-up care was 9 months (range, 3-29 months). Primary union was achieved in all fractures. The average range of motion was 83 degrees for the MP joint and 66 degrees for the proximal interphalangeal joint. The average total active motion (TAM) for the involved digits was 200 degrees (range, 65 degrees-250 degrees). Fractures without intra-articular extension had a significantly better average TAM (213 degrees) than did those with intra-articular extension (169 degrees; p = .05). Primary bone grafting did not adversely effect the final TAM. There were no infections. Early stable fracture fixation of these injuries achieved union, alignment, and early rehabilitation with no appreciable increase in morbidity.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Multiple attachment mechanisms of corneal epithelial cells to a polymer--cells can attach in the absence of exogenous adhesion proteins through a mechanism that requires microtubules

STUDY OBJECTIVE: To determine whether emergency patients with acute chest pain and low suspicion of acute myocardial infarction (AMI) can be managed cost-effectively and safely in a dedicated chest pain center (CPC) that incorporates mandatory stress testing. METHODS: We assembled a prospective observational case series of consecutive adult patients transferred from the emergency department to a nine-bed, 23-hour CPC in a 564-bed community hospital from January 13 through May 31, 1994. In our institution, all emergency patients with acute nontraumatic chest pain of unclear origin, suggestive of myocardial ischemia but with a low probability of AMI, are transferred to the CPC for further evaluation. All patients in whom AMI is ruled out undergo individually appropriate cardiac diagnostic testing in accordance with CPC clinical guidelines. Patients with end-stage coronary artery disease transferred to the CPC for a "rule-out" protocol only did not undergo further diagnostic testing. Admitted and discharged patients were followed through chart review and telephone survey, respectively. RESULTS: Of the 502 patients transferred to the CPC, 477 (95%) completed follow-up at 14 days. Four hundred ten (86%) were discharged home. Those discharged after diagnostic evaluation yielded negative findings had 100% survival and zero diagnosis of AMI at 5-month follow-up. Overall mortality and incidence of AMI on long-term follow-up for all patients transferred to the CPC were .4% and .2%, respectively. Sixty-seven patients (13%) were admitted from the CPC, of whom 44 (66%) had a final diagnosis of ischemic heart disease (IHD) or AMI. Twenty-four patients with IHD (55%; 6% of stress-tested group) were identified only on further stress testing. Of these patients, seven underwent percutaneous transluminal coronary angioplasty or coronary artery bypass grafting during hospitalization. All were discharged home without major morbidity. Four hundred twenty-four patients (84%) underwent stress testing. The cost of mandatory stress testing to identify one patient with IHD after AMI was ruled out was $3,125. An average cost-per-case savings of 62% was achieved for each patient transferred to the CPC who would have been hospitalized before the inception of the CPC. CONCLUSION: Mandatory stress testing is a safe, cost-effective, and valuable diagnostic and prognostic tool in CPC patients.     

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.     

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.