Knockdown of OLR1 weakens glycolytic metabolism to repress colon cancer cell proliferation and chemoresistance by downregulating SULT2B1 via c-MYC

Chemoresistance is one of the major problems of colon cancer treatment. In tumors, glycolytic metabolism has been identified to promote cell proliferation and chemoresistance. However, the molecular mechanisms underlying glycolytic metabolism and chemoresistance in colon cancer remains enigmatic. Hence, this research was designed to explore the mechanism underlying the OLR1/c-MYC/SULT2B1 axis in the regulation of glycolytic metabolism, to affect colon cancer cell proliferation and chemoresistance. Colon cancer tissues and LoVo cells were attained, where OLR1, c-MYC, and SULT2B1 expression was detected by immunohistochemistry, RT-qPCR, and western blot analysis. Next, ectopic expression and knockdown assays were implemented in LoVo cells. Cell proliferation was detected by MTS assay and clone formation. Extracellular acidification, glucose uptake, lactate production, ATP/ADP ratio, and GLUT1 and LDHA expression were measured to evaluate glycolytic metabolism. Then, the transfected cells were treated with chemotherapeutic agents to assess drug resistance by MTS experiments and P-gp and SMAD4 expression by RT-qPCR. A nude mouse model of colon cancer transplantation was constructed for in vivo verification. The levels of OLR1, c-MYC, and SULT2B1 were upregulated in colon cancer tissues and cells. Mechanistically, OLR1 increased c-MYC expression to upregulate SULT2B1 in colon cancer cells. Moreover, knockdown of OLR1, c-MYC, or SULT2B1 weakened glycolytic metabolism, proliferation, and chemoresistance of colon cancer cells. In vivo experiments authenticated that OLR1 knockdown repressed the tumorigenesis and chemoresistance in nude mice by downregulating c-MYC and SULT2B1. Conclusively, knockdown of OLR1 might diminish SULT2B1 expression by downregulating c-MYC, thereby restraining glycolytic metabolism to inhibit colon cancer cell proliferation and chemoresistance.Subject terms: Cancer, Cancer therapy     

Combined Fourier transform infrared and Raman spectroscopic approach for identification of multidrug resistance phenotype in cancer cell lines

Cancer cells escape cytotoxic effects of anticancer drugs by a process known as multidrug resistance (MDR). Identification of cell status by less time-consuming methods can be extremely useful in patient management and treatment. This study aims at evaluating the potentials of vibrational spectroscopic methods to perform cell typing and to differentiate between sensitive and resistant human cancer cell lines, in particular those that exhibit the MDR phenotype. Micro-Raman and Fourier transform infrared (FTIR) spectra have been acquired from the sensitive promyelocytic HL60 leukemia cell line and two of its subclones resistant to doxorubicin (HL60/DOX) and daunorubicin (HL60/DNR), and from the sensitive MCF7 breast cancer cell line and its MDR counterpart resistant to verapamil (MCF7/VP). Principal components analysis (PCA) was employed for spectral comparison and classification. Our data show that cell typing was feasible with both methods, giving two distinct clusters for HL60- and MCF7-sensitive cells. In addition, phenotyping of HL60 cells, i.e., discriminating between the sensitive and MDR phenotypes, was attempted by both methods. FTIR could not only delineate between the sensitive and resistant HL60 cells, but also gave two distinct clusters for the resistant cells, which required a two-step procedure with Raman spectra. In the case of MCF7 cell lines, both the sensitive and resistant phenotypes could be differentiated very efficiently by PCA analysis of their FTIR and Raman point spectra. These results indicate the prospective applicability of FTIR and micro-Raman approaches in the differentiation of cell types as well as characterization of the cell status, such as the MDR phenotype exhibited in resistant leukemia cell lines like HL60 and MCF7.     

Identification of molecular targets for selective elimination of TRAIL‐resistant leukemia cells. From spots to in vitro assays using TOP15 charts

The resistance of malignant cells to chemotherapy calls for the development of novel anti‐cancer drugs. TNF‐related apoptosis‐inducing ligand (TRAIL) is a pro‐apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL‐resistant HL‐60 subclones, HL‐60/P1 and HL‐60/P2, from a TRAIL‐sensitive HL‐60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable “weaknesses” of the TRAIL‐resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2‐DE) analysis and compared both TRAIL‐resistant subclones with the original TRAIL‐sensitive HL‐60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so‐called “TOP15” proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL‐60/P1 cells, and the marked down‐regulation of enzyme adenosine deaminase in HL‐60/P2 cells, suggests increased sensitivity of these cells to DNA‐interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL‐60/P1 cells, and adenosine and vidarabine to HL‐60/P2, compared with TRAIL‐sensitive HL‐60 cells.     

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3–4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage in the of VP-16 was, in part, related to a 2–3-fold decrease in both the amount and activity of topisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

CXCL2/CXCR2 axis induces cancer stem cell characteristics in CPT-11-resistant LoVo colon cancer cells via Gαi-2 and Gαq/11

Cancer stem cells (CSCs) exist in colon cancer and exhibit characteristics of stem cells which are due to lineages of tissues where they arise. Epithelial to mesenchymal transition (EMT)-undergoing cancer cells display CSC properties and therapeutic resistance. Cancer and stromal cells comprise of a tumor microenvironment. One way the two populations communicate with each other is to secret CXC ligands (CXCLs). CXCLs are capable of causing chemotaxis of specific types of stromal cells and control angiogenesis. Double immunofluorescence, western blot analysis, and colony-formation assay were carried out to compare parental and CPT-11-resistant LoVo cells. CPT-11-R LoVo colon cancer cells showed increased expression of CXCL1, CXCL2, CXCL3, and CXCL8. They displayed significantly increased intracellular protein levels of CXCL2 and CXCR2. CPT-11-R LoVo cells showed significantly elevated expression in aldehyde dehydrogenase 1 (ALDH1), cluster of differentiation 24 (CD24), cluster of differentiation 44 (CD44), and epithelial cell adhesion molecule (EpCAM). CXCL2 knockdown by short hairpin RNA resulted in reduced expression of CSC proteins, cyclins, EMT markers, G proteins, and matrix metalloproteinases (MMPs). Finally, Gαi-2 was found to promote expression of CSC genes and tumorigenesis which were more apparent in the resistant cells. In addition, Gαq/11 showed a similar pattern with exceptions of EpCAM and MMP9. Therefore, CXCL2–CXCR2 axis mediates through Gαi-2 and Gαq/11 to promote tumorigenesis and contributes to CSC properties of CPT-11-R LoVo cells.     

Exposure of human leukemic cells to direct electric current

Treatment with direct electric current (DC) influences the growth of several cancer cells. In this work, we evaluated the effects of DC treatment on the human leukemic cell line HL60. Human cells were separately treated in the presence of the cathode or the anode or without contact with the electrodes. In all systems, DC-treated cells presented an impaired ability to proliferate. Growth inhibition was dependent on the generation of soluble products of electrolysis. Cathodic treatment of HL60 cells predominantly induced lysis, whereas treatment without contact with electrodes did not induce alterations in cell viability. In contrast, cell stimulation by the anode resulted in irreversible membrane damage, as demonstrated by trypan blue and 7-aminoactinomycin staining. Analysis of these cells by transmission electron microscopy indicated that necrosis is a major mechanism inducing cell death. In addition, apoptotic-like cells were observed under light microscopy after anodic treatment. Accordingly, DNA from anodic-treated cells presented a typical pattern of apoptosis. Apoptotic cell death was only generated after the treatment of HL60 cells in conditions in which the generation of chloride-derived compounds was favored. These results indicate that the nature of the products from cathodic or anodic reactions differently influences the mechanisms of cell death induced by DC-derived toxic compounds.     

Dimethylsulfoxide, retinoic acid and 12-O-tetradecanoylphorbol-13-acetate induce a selective decrease in the phosphorylation of P150, a surface membrane phosphoprotein of HL60 cells resistant to adriamycin

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors.

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

Pyruvate dehydrogenase kinase 1 contributes to cisplatin resistance of ovarian cancer through EGFR activation

Patients with ovarian cancer frequently develop acquired drug resistance after the long-term chemotherapy, leading to disease progression. Enhanced epithelial–mesenchymal transition (EMT) has been implicated in chemoresistance of ovarian cancer cells; however, the molecular mechanisms involved are largely undefined. Pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, has been recognized as a gatekeeper of the Warburg effect, a hallmark of cancer. In this study, the function of PDK1 in cisplatin resistance of ovarian cancer in terms of growth and EMT was investigated. PDK1 was upregulated in cisplatin-resistant ovarian cancer cells. PDK1 knockdown in resistant cells led to increased sensitivity to cisplatin-induced cell death and apoptosis. PDK1 downregulation also reversed the EMT and cell motility in cisplatin-resistant cells. In a mouse xenograft model, tumors derived from PDK1-silenced ovarian cancer cells exhibited decreased tumor growth and EMT compared with control after the cisplatin treatment. Mechanistically, PDK1 overexpression led to increased phosphorylation of EGFR, and blocking EGFR kinase activity by erlotinib reversed cisplatin resistance induced by PDK1 overexpression. Furthermore, in patients with ovarian cancer, higher PDK1 and p-EGFR levels were associated with chemoresistance. These results supported that PDK1 contributes to chemoresistance of ovarian cancer by activating EGFR. Therefore, PDK1 may serve as a promising target to combat chemoresistance of ovarian cancer.     

Mechanisms of resistance to the cytotoxic effects of oxysterols in human leukemic cells

We have developed hematopoietic cells resistant to the cytotoxic effects of oxysterols. Oxysterol-resistant HL60 cells were generated by continuous exposure to three different oxysterols-25-hydroxycholesterol (25-OHC), 7-beta-hydroxycholesterol (7beta-OHC) and 7-keto-cholesterol (7kappa-C). We investigated the effects of 25-OHC, 7beta-OHC, 7kappa-C and the apoptotic agent staurosporine on these cells. The effect of the calcium channel blocker nifedipine on oxysterol cytotoxicity was also investigated. Differential display and real-time PCR were used to quantitate gene expression of oxysterol-sensitive and -resistant cells. Our results demonstrate that resistance to the cytotoxic effects of oxysterols is relatively specific to the type of oxysterol, and that the cytotoxicity of 25-OHC but not that of 7beta-OHC and 7kappa-C, appears to occur by a calcium dependent mechanism. Oxysterol-resistant cells demonstrated no significant difference in the expression of several genes previously implicated in oxysterol resistance, but expressed the bcl-2 gene at significantly lower levels than those observed in control cells. We identified three novel genes differentially expressed in resistant cells when compared to HL60 control cells. Taken together, the results of this study reveal potentially novel mechanisms of oxysterol cytotoxicity and resistance, and indicate that cytotoxicity of 25-OHC, 7beta-OHC and 7kappa-C occur by independent, yet overlapping mechanisms.     

miR-1275 targets MDK/AKT signaling to inhibit breast cancer chemoresistance by lessening the properties of cancer stem cells

Chemoresistance is a major obstacle in the neoadjuvant chemotherapy (NCT) of locally advanced breast cancer (LABC). Identification of miRNAs as prognostic biomarkers may help overcome chemoresistance of breast cancer (BC). This study aimed to evaluate the expression level of miR-1275 in plasma samples and its biological functions in the chemoresistance of BC. The expression levels of miR-1275 in plasma samples and cells were measured by RT-qPCR. CRISPR/Cas9-mediated gene editing was used to construct miR-1275 knock-out cells in MCF-7. We found that miR-1275 was significantly downregulated in plasma from patients resistant to chemotherapy and in chemoresistant BC cell lines, while patients with low levels of miR-1275 showed poor overall survival. miR-1275 knock-out promoted chemoresistance in BC cells by increasing the properties of cancer stem cells (CSCs). Mechanistically, we identified that MDK was determined to be direct downstream protein of miR-1275 which initiated PI3K/Akt signaling in breast cancer cells. We demonstrated that the high expression level of miR-1275 in plasma predicted better response to NCT. The reduction of miR-1275 promoted BC cells chemoresistance by increasing CSCs properties via targeting MDK/AKT axis. The potential of miR-1275 as a new prognostic biomarker and therapeutic target of BC patients was identified.     

Proteomics for identifying mechanisms and biomarkers of drug resistance in cancer

A major problem in chemotherapy of cancer patients is drug resistance as well as unpredictable response to treatment. During chemotherapy, multiple alterations of genetics and epigenetics that contribute to chemoresistance take place, eventually impacting on disease outcome. A more complex picture of the mechanisms of drug resistance is now emerging through application of high-throughput proteomics technology. We have entered an exciting time where proteomics are being applied to characterize the mechanisms of drug resistance, and to identify biomarkers for predicting response to chemotherapy, thereby leading to personalized therapeutic strategies of cancer patients. Comparative proteomics have identified a large number of differentially expressed proteins associated with chemoresistance. Although roles and mechanisms of such proteins in chemoresistance need to be further proved, at least some of them may be potential biomarkers for predicting chemotherapeutic response. Herein, we review the recent advancements on proteomic investigation of chemoresistance in human cancer, and emphasize putative biomarkers for predicting chemotherapeutic response and possible mechanisms of chemoresistance identified through proteomic approaches. Suggested avenues for future work are discussed.     

Anticancer and superoxide scavenging activities of p-alkylaminophenols having various length alkyl chains

A series of p-alkylaminophenols including 3, p-butylaminophenol; 4, p-hexylaminophenol; 5, p-octylaminophenol; and 6, N-(p-methoxybenzyl)aminophenol were synthesized based on the structure of fenretinide, N-(4-hydroxyphenyl)retinamide (1). This latter agent is a synthetic amide of all-trans-retinoic acid (RA), which is a cancer chemopreventive and antiproliferative agent. It was found that elongation of the alkyl chain length in these compounds increased antioxidative activity and inhibition of lipid peroxidation. These findings led us to investigate whether antiproliferative activity against cancer cells was effected by the length of alkyl chains linked to the aminophenol residue. All p-alkylaminophenols inhibited growth of HL60 and HL60R cells in a dose-dependent manners. The HL60R line is a resistant clone against RA. Growth of various cancer cell lines (HL60, HL60R, MCF-7, MCF-7/Adr(R), HepG2, and DU-145) was suppressed by p-alkylaminophenols in a fashion dependent on the aminophenol alkyl chain length (5>4>3>p-methylaminophenol (2)), with 5 being the most potent inhibitor of cell growth against HL60R, MCF-7/Adr(R), and DU-145 cells among p-alkylaminophenols tested, including 1. In particular, with the exception of compound 2, antiproliferative activity against DU-145 cells by these p-alkylaminophenols was greater than by 1. In HL60 cells, growth inhibition was associated with apoptosis. On the other hand, elongation of the alkyl chain length reduced superoxide trapping capability (2>3>4>5) in contrast to the effects on inhibition of lipid peroxidation. These results indicate that anticancer activity of p-alkylaminophenols correlated with the inhibitory activity of lipid peroxidation, but not with the superoxide scavenging activity.     

Potentiated DNA Damage Response in Circulating Breast Tumor Cells Confers Resistance to Chemotherapy

Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis.     

Specific fluorescent tracers. Imaging and applications for photodynamic therapy

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy. In addition, we will present two additional routes, whose aims are to create new features to respond to the PDT questioning: firstly, the synthesis of fluorescent tracers, with a particular attention to the presence of hydrophilic groups (glucosamine ring) on the basic fluorophore structure to orientate the localisation of the probe and, secondly, the use of scanning near-field optical microscopy to reach a better resolution for the fluorescence microscopy analysis.     

Adriamycin resistance in HL60 cells in the absence of detectable P-glycoprotein

Previous studies have shown that the development of multi-drug resistance in cell lines treated with chemotherapeutic agents is closely associated with the overexpression of a 170-180 kilodalton surface membrane glycoprotein (P-glycoprotein). In the present study a monoclonal antibody against the P-glycoprotein was used to determine if this protein is overexpressed in multi-drug resistant HL60 cells. Using either indirect immunofluorescent staining or immunoblot analysis P-glycoprotein could not be detected in HL60 cells isolated for resistance to adriamycin. In contrast HL60 cells isolated for resistance to vincristine contain the P-glycoprotein and the amount of this material increases with increasing levels of resistance. These studies thus demonstrate adriamycin resistance in P-glycoprotein negative HL60 cells. Furthermore adriamycin and vincristine are found to have distinct effects in inducing overexpression of P-glycoprotein in the HL60 cell line. This information could be useful in the development of therapeutic strategies for the treatment of certain forms of cancer.     

Retinoylation of proteins in leukemia, embryonal carcinoma, and normal kidney cell lines: differences associated with differential responses to retinoic acid

In HL60 cells a nuclear protein of Mr 55,000 is retinoylated, with the formation of a thioester bond. To gain further knowledge on the role of retinoylation we studied it in cell lines with varied responses to retinoic acid (RA). Compared to HL60 the extent of retinoylation (mol/cell) was about fivefold higher in HL60/MRI, a mutant which is more sensitive to RA than HL60. Retinoylation occurred to the same extent and at similar rates in HL60 and in HL60/RA-res, a mutant resistant to differentiation by RA. One-dimensional polyacrylamide gel electrophoresis patterns for the three HL60 cell lines were similar. However, two-dimensional polyacrylamide gel electrophoresis patterns of the three HL60 cell lines were distinct. While we saw the same major retinoylated protein of Mr 55,000 in the three cell lines, the HL60/RA-res cells also contained a high level of a protein with the same Mr and a lower pI. The extent of retinoylation was greater in the RA-sensitive embryonal carcinoma cell line, PCC4.aza1R, than in a RA-resistant cell line, PCC4.(RA)-2. One-dimensional polyacrylamide gel electrophoresis patterns of retinoylated proteins of the embryonal carcinoma cell lines were different from HL60 and from each other. The retinoylation pattern of the normal canine kidney cell line (MDCK) was different from either HL60 or the embryonal carcinoma cells. These results showed the retinoylation was widespread and that the response to RA of different cell types may depend on the retinoylation of specific proteins.