ras-transformed cells: altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites.

Steady-state cellular levels of phosphatidylinositol-4,5-bisphosphate (PIP2), 1,2-diacylglycerol (DAG), and inositol phosphates have been measured in two different fibroblast cell lines (NIH 3T3 and NRK cells) before and after transformation with three different ras genes. At high cell density the ratio of DAG to PIP2 was 2.5- to 3-fold higher in the ras-transformed cells than in their untransformed counterparts. The sum of the water-soluble breakdown products of the polyphosphoinositides, inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate, was also elevated in ras-transformed NRK cells compared with nontransformed NRK cells. These findings suggest that the ras (p21) protein may act by affecting these levels, possibly as a regulatory element in the PIP2 breakdown pathway.

Immunohistochemical localization of apolipoprotein E in human glial neoplasms.

Immunocytochemical analyses revealed the presence and distribution of apolipoprotein E (apo E) in normal human brain tissue as well as in 77 human intracranial neoplasms. In normal brain tissues, the perikarya of astrocytes exhibited a strong positive reaction, whereas the Bergmann glia were stained to a moderate degree. However, no immunoreactivity was observed with neurons, oligodendrocytes, ependymal cells, and choroidal epithelium. Among the intracranial neoplasms, oligodendroglioma, choroid plexus papilloma, hemangioblastoma, primary malignant lymphoma, neurinoma, meningioma, pituitary adenoma, and craniopharyngioma were all negative. Immunoreactivity in the peripheral neuroblastoma was nil. However, the perikarya of astrocytomas and glioblastomas showed a positive reaction. Analyses on the degree of anaplasia and the amount of apo-E as an intensity of immunostaining showed a negative correlation. The astrocytic elements were stained in mixed oligoastrocytomas and medulloblastomas with glial differentiation. A few cases of ependymomas showed weak perikaryal immunostaining. Western blot analyses with anti-apo E antibody of a freshly prepared surgical specimen with astrocytomas revealed a single band with a molecular weight of approximately 37,000. The well differentiated cultured human astrocytoma cells secreted apo E into the medium. These lines of evidence suggest that apo E may serve as a potential marker specific for astrocytomas and glioblastomas, as well as an indicator of astrocytic tumor cell differentiation. The apo E localization in human brain tumors could be clinically relevant and diagnostically useful.

L-myc cooperates with ras to transform primary rat embryo fibroblasts.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

Purification of a gelatin-degrading type IV collagenase secreted by ras oncogene-transformed fibroblasts.

Connective tissue matrix-degrading metalloproteinases play an important role in cancer invasion. In this report we describe the isolation of a metalloproteinase exhibiting both type IV collagenolytic and gelatinolytic activities from the conditioned medium of NIH-3T3 fibroblasts transformed with DNA containing an activated c-Harvey-ras oncogene from T24 bladder cancer cells. This tumor proteinase was purified by anion exchange chromatography, zinc-chelate Sepharose chromatography, and gel permeation chromatography. The final product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (relative molecular mass = 67,000). Gelatin zymography revealed two bands of gelatinolytic activity, corresponding to molecular weights of 67,000 and 62,000. Upon immunoblotting with the use of an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase that lacks homology with interstitial collagenase or stromelysin, the purified tumor enzyme was identified as type IV collagenase.

Detection of a tumor-associated nucleoprotein antigen in mink lung cells transformed by two different viral oncogenes.

Salt-precipitated chromatin was prepared from cultured MvlLu line mink lung cells and from these cells transformed by either of the oncogenes v-mos (MIMS-102 line) or v-fes (F3C7 line). Xenoantisera were raised to chromatin from each of the three cell types and cross-tested in microcomplement fixation assays to determine immunospecificity. Chromatin from cells transformed by either v-mos or v-fes revealed antigenic profiles statistically indistinguishable (P less than 0.2 to 0.5) from one another with their respective cross-tested antisera, but did not react significantly with antisera to chromatin from the untransformed parental cell line. Likewise, little cross-reaction was observed with chromatin from the untransformed cells and antisera raised to chromatin from either of the oncogenically transformed lines (P less than 0.001), although each chromatin demonstrated high reactivity with its homologous antiserum preparation. These immunological data are consistent with the observed normal or transformed characteristics for each cell type, including morphology, anchorage-independent growth, and growth in the absence of serum.

Spontaneous neoplastic evolution of Chinese hamster cells in culture: multistep progression of karyotype.

Chromosomes from successive passages of a Chinese hamster cell strain (WCHE/5) that spontaneously progressed from a euploid primary cell culture to a heteroploid tumorigenic cell line were isolated and analyzed by Giemsa banding and high-resolution flow karyotype analysis. The frequency and identification of aneuploid and marker chromosomes were determined at both pre- and postcrisis culture stages and pre- and posttumorigenic stages. The combination of Giemsa banding and flow karyotypes provided detailed analysis of karyotype instability at each stage of cell culture progression. Aneuploidy (trisomy of chromosome 5) preceded the appearance of tumorigenicity in nude mice as well as in vitro indicators of neoplasia. The four stages of neoplastic progression defined in the previous paper correlated with a steady progression in karyotypic instability, including, in sequence: trisomy of chromosome 5; an 8q marker chromosome; a 3q+ insertion; and trisomy of chromosome 8. Additional changes continued to appear as the cells acquired classical properties of in vitro transformation.

Evidence for multiple steps in neoplastic transformation of normal and preneoplastic Syrian hamster embryo cells following transfection with Harvey murine sarcoma virus oncogene (v-Ha-ras).

Neoplastic development of Syrian hamster embryo (SHE) cells in culture is a multistep process in which intermediate or preneoplastic cells can be identified and isolated. In an attempt to further characterize normal and preneoplastic cells, we have compared their susceptibilities to neoplastic transformation following transfection with cloned DNA of the oncogenic virus, Harvey murine sarcoma virus (HaMSV). Normal SHE cells, which are stably nontumorigenic when injected in nude mice, are competent to take up and express exogenous DNA as demonstrated by transfection experiments with pSV2-neo DNA and certain viral DNAs. SHE cells treated with 5 micrograms of HaMSV DNA per dish remained nontumorigenic. Colonies of SHE cells, isolated after cotransfection with HaMSV and pSV2-neo DNA and selection for G418 antibiotic resistance, expressed Harvey murine sarcoma virus oncogene (v-Ha-ras) RNA and were initially morphologically altered; however, all colonies senesced when subcultured. In contrast, transfection of the cells with polyoma virus DNA alone or HaMSV DNA plus MC29 viral DNA (pSVv-myc) and then injection of the cells into nude mice resulted in progressively growing tumors of hamster origin within 3 to 5 weeks. A preneoplastic cell line, DES-4, isolated after treatment of SHE cells with the human carcinogen diethylstilbestrol, was chosen for comparative analyses. These immortalized cells are nontumorigenic and excellent recipients for exogenous DNA. In contrast to SHE cells, DES-4 cells were highly susceptible to neoplastic transformation following transfection with HaMSV DNA. To further investigate the role of HaMSV DNA in the neoplastic transformation of DES-4 cells and to determine whether this occurred as a single step, clones of DES-4 cells cotransfected with pSV2-neo and HaMSV DNAs were selected by antibiotic resistance and characterized. There was a good correlation between tumorigenicity and expression of v-Ha-ras DNA; however, the clones were highly variable in terms of their latency periods in vivo and anchorage-independent growth. Neither of these two parameters correlated with the level of expression of v-Ha-ras RNA. All of the cell lines derived from tumors and reinoculated into nude mice had short latency periods in vivo, were highly anchorage independent, and had high levels of v-Ha-ras expression. These results suggest that, in these experiments, v-Ha-ras expression was necessary, but not sufficient, for the tumorigenicity of DES-4 cells and that additional changes in the cells were acquired.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of neoplasia-related proteins of chemically transformed HuT fibroblasts in human osteosarcoma HOS fibroblasts and modulation of actin expression upon elevation of tumorigenic potential.

Two sets of abundant cytoplasmic transformation-specific polypeptides, p788/p789 and p219/p220, have been identified by comparing in vitro-transformed human fibroblasts with diploid human fibroblasts. These polypeptides are also expressed by the human fibrosarcoma and osteosarcoma cell lines HT1080 the human fibrosarcoma and osteosarcoma cell lines HT1080 and HOS, respectively. HOS cells, however, synthesize only one of the two electrophoretic forms of each marker set, p789 and p219, at greatly reduced rates compared to the rates of synthesis found for HT1080 cells and the in vitro-transformed cell lines. Induction of expression of these neoplastic marker polypeptides is independent of the activation of a transforming gene that will induce focus formation in confluent mouse 3T3 cell monolayers. Activation of the met oncogene in MNNG-HOS cells and simultaneous elevation of tumorigenic potential did not lead to a significant change in the rate of the 600 most abundant polypeptide species with the exception of one of the two cytoplasmic actin polypeptides. While the normal ratio of beta-to gamma-actin which is approximately 2:1 was expressed in "untransformed" HOS cells, MNNG-HOS cells synthesized 50% less beta-actin resulting in a 1:1 ratio of beta-actin to gamma-actin. Our finding here, together with our previous characterization of the human beta-actin gene, leads us to predict that one of two functional beta-actin genes expressed in HOS cells has been inactivated in MNNG-HOS cells by either a regulatory or structural gene mutation.

Reexpression of fetal characters in simian virus 40-transformed human keratinocytes.

Normal human keratinocytes are able to stratify, form cornified squames, and terminally differentiate in tissue culture. These properties are frequently impaired by malignant transformation. In the present paper, we show that, in addition, transformation by SV40 results in the coordinate reexpression of a whole set of fetal characters. Moreover, a comparison of two SV40-transformed human keratinocyte cell lines, one still showing a certain degree of stratification and terminal differentiation (HE-SV) and the other almost completely unable to differentiate (SVK14), suggests that the impairment of differentiation and the intensity of reexpression of fetal markers are correlated. Particularly, a set of three keratin polypeptides, absent in adult stratified epithelia but normally found in the nonstratified fetal epidermis, is present in much larger amounts in SVK14 cells than in HE-SV cells. On the other hand, the inability of SVK14 cells, in contrast to HE-SV cells, to form cornified envelopes seems to be due to the inability of those cells to accumulate involucrin.

Organization and expression of endogenous virus-like (VL30) DNA sequences in nontransformed and chemically transformed mouse embryo cells in culture.

A cloned mouse DNA fragment containing an endogenous "virus-like" DNA (VL30 DNA) sequence was identified by virtue of its ability to hybridize to the virus-like RNA component of mixed-pseudotype AKR-murine leukemia virus virions, its lack of detectable sequence homology with cloned AKR-murine leukemia virus DNA, and its hybridization to a 5.6 kilobase pair (30S) cellular polyadenylic acid [poly(A)]-containing RNA species. Restriction enzyme mapping of the cloned mouse fragment revealed the presence of a 5- to 6-kilobase pair VL30 DNA segment flanked by non-VL30 segments of approximately 7 and 0.3 kilobase pairs. Southern blot analysis of VL30 DNA sequence organization in the DNA of two nontransformed mouse cell lines (AKR-2B, C3H/10T 1/2) and two chemically transformed derivatives (AKR-MCA, C3H/MCA-58) revealed 15 to 20 bands organized in an apparent strain-specific pattern. Within a given strain, however, no differences were detectable between the nontransformed cells and their chemically transformed counterparts. The expression of VL30 genes in the above cell lines was assayed by hybridization of 32P-labeled poly(A)-containing polysomal RNA to several internal restriction fragments derived from the cloned VL30 DNA sequence. The level of VL30 RNA was enhanced approximately 10-fold in both chemically transformed cell lines as compared to the nontransformed cell lines (under normal growth conditions). In addition, nontransformed AKR-2B cells maintained in the presence of purified epidermal growth factor exhibited similarly enhanced levels of VL30 RNA sequences in polysomal RNA. Since these cells displayed several growth characteristics of transformed cells but, in an epidermal growth factor-dependent and completely reversible fashion, these data suggest that the expression of VL30 genes is not simply incidental to chemically transformed cells but may be related to alterations in growth control.

Cell surface features associated with differentiation-induction of methylcholanthrene-transformed AKR-2B fibroblasts by N,N-dimethylformamide.

Methylcholanthrene-transformed AKR-2B mouse embryonal fibroblasts (AKR-MCA cells) were examined for cell surface alterations after growth in culture medium containing N,N-dimethylformamide (DMF) using the lactoperoxidase-glucose oxidase radioiodination procedure with subsequent electrophoresis. DMF has been shown to induce maturational changes in a variety of transformed cells in vitro and has been reported to produce a more normal phenotype when applied to cultured AKR-MCA cells. The electrophoretic profile of 125I-labeled surface proteins from AKR-MCA cells exhibited a prominent peak of labeled material with a molecular weight of approximately 85,000. After growth of AKR-MCA cells in medium containing DMF, the Mr 85,000 peak was substantially reduced, while there was a large increase in Mr 200,000 to 250,000 radioiodinated surface material. This cell surface labeling pattern was virtually identical to that of the nontransformed AKR-2B fibroblasts from which AKR-MCA cells were derived. The cell surface alterations observed upon exposure of AKR-MCA cells to DMF occurred as a function of time of growth in DMF and DMF concentration. Growth of AKR-MCA cells in DMF resulted in a steady increase in cell surface 125I incorporation up to the fourth day of exposure to DMF. At this time, the incorporation level was 22.9-fold greater than that for untreated AKR-MCA cells. Incorporation of radiolabel was decreased after the fifth and sixth days of AKR-MCA exposure to DMF. This trend was also manifested by AKR-2B fibroblasts grown in the presence of DMF. The data suggest that there was increased expression of the Mr 200,000 to 250,000 surface protein on both AKR-2B and AKR-MCA cells when grown in DMF. DMF also inhibited morphological transformation and the cell surface changes associated with transformation of AKR-2B cells by exogenous transforming growth factors.

Purification and biological properties of type beta transforming growth factor from mouse transformed cells.

Transforming growth factor type beta (TGF beta) has been purified from serum-free culture fluids of transformed mouse L-929 cells which are capable of continual growth in serum-free medium in the absence of any exogenously added polypeptide growth factors. TGF beta has been purified to homogeneity as judged by NH2-terminal amino acid sequence analysis. Analysis of the purified polypeptide by gel electrophoresis indicates that TGF beta is composed of two polypeptide chains of Mr 12,500 cross-linked by disulfide bonds. TGF beta was characterized by its ability to induce anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar in the presence of epidermal growth factor (EGF). TGF beta was also able to enhance both EGF-induced DNA synthesis and cell proliferation on growth-arrested NRK cells in monolayer cultures under serum-free conditions. We also show that in mouse melanoma B-16 cells under serum-free conditions TGF beta stimulates both DNA synthesis in monolayer cultures and anchorage-independent growth in soft agar. Paradoxically, the anchorage-independent growth in the presence of serum of many human cell lines, including melanomas, and mammary, prostatic, vulvar, and lung carcinomas is inhibited by TGF beta at saturating concentrations similar to those that stimulate colony formation of the rodent cell lines L-929 and B-16 under serum-free conditions. The peculiar action of TGF beta is further revealed by the observations that while EGF and TGF beta synergize to induce inhibition of anchorage-independent growth of A-431 human vulvar carcinoma cells, their effects on the anchorage-independent growth of one human lung carcinoma cell line (A-549) and two human prostatic carcinoma cell lines (PC-3 and DU-145) are antagonistic. Moreover, we show that in the rodent and human cell lines TGF beta interacts with specific cellular receptors which may mediate the actions of TGF beta. We conclude that the expression of both TGF beta and TGF beta receptors by L-929 cells and the stimulation of growth of L-929 cells in serum-free medium by TGF beta suggests that TGF beta may be important for maintaining the transformed state of this tumor cell line, and the way in which a cell responds to TGF beta is dependent on the presence or absence of growth factors contained in the serum.

Hepatoma-associated nonhistone chromosomal proteins.

In order to compare nonhistone proteins in normal and neoplastic hepatocytes, we elicited antisera to Morris hepatoma 7777 dehistonized chromatin. By the enzyme-linked immunosorbent assay, the antisera demonstrated specificity for Morris hepatoma 7777 and little reactivity to normal rat liver chromatin. Morris hepatomas 7288c, 7800 and 5123tc shared some antigenic hepatoma nonhistone proteins. Neoplasia induced in rats fed 3'-methyl-4-dimethylaminoazobenzene changed the immunospecificity of the liver chromatin to a new type that was antigenically similar to Morris hepatoma 7777. Fetal rat liver chromatin and regenerating rat liver chromatin did not bind antibody. To further characterize the antigenic nonhistone proteins, we analyzed Morris hepatoma 7777 chromatin and normal rat liver chromatin by the immunoblot technique. Nonhistone proteins that demonstrated immunoreactivity were predominantly high molecular weight proteins.

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.

Detection of tumor necrosis factor gene expression at a cellular level in human acute myeloid leukemias.

Tumor necrosis factor (TNF) is a Mr 17,000 cytokine produced by macrophages. We have recently demonstrated that TNF is also produced by transformed human epithelial cells. The present studies have examined TNF expression in human myeloid leukemic cells. We have monitored TNF expression at a cellular level using alkaline phosphatase detection of a biotinylated TNF cDNA probe in situ. Using this approach, TNF transcripts were detectable in HL-60 cells induced along the monocytic lineage by phorbol ester but not in uninduced cells. The specific detection of TNF RNA at a cellular level was supported by the absence of histochemical staining in RNase-treated cells and when using biotinylated pBR322 plasmid without insert. These studies were extended to preparations of purified acute myeloblastic leukemia cells. The results demonstrate that TNF is expressed in myeloblasts in eight of nine patients with AML. In each preparation of myeloblasts with detectable TNF RNA, transcripts were present at 89-98% of the cells. The identification of TNF RNA in situ was also associated with the detection of TNF protein in leukemic blasts by indirect immunofluorescence. Moreover, the detection of TNF protein in these preparations of myeloblasts was confirmed by immunoblotting. However, using this approach to examine AML cells before and after purification indicated that TNF expression is induced as a result of the enrichment procedures. Thus, certain populations of purified myeloid leukemic cells are capable of expressing TNF at both the RNA and protein levels.

T cell receptor alpha mRNA transcription in T-lymphoblastic transformation of chronic myelocytic leukemia.

Analysis at the DNA and RNA level revealed a mature genetic marker profile in a case of T type blast crisis of chronic myelocytic leukemia. T cell receptor beta chain gene rearrangement as well as T cell receptor alpha mRNA transcription was demonstrated in blasts of the malignant clone. Corresponding findings were obtained from immunological phenotyping. Blasts were found to be CD7+, CD1+, CD3+, CD4+, CD8+, and TdT- and classified as common/mature thymocytes. The presence of the breakpoint cluster region gene on chromosome 22 excluded the possibility of a second neoplastic process.

Gene rearrangements of T cell receptor beta and gamma chains in HTLV-I infected primary neoplastic T cells.

Rearrangements of T cell receptor beta and gamma chain (T beta and T gamma) genes were analyzed by Southern blot method in samples from 30 patients with adult T cell leukemia (ATL) and 17 patients with non-ATL T cell neoplasms. The DNA probes used were the constant and joining region of T beta gene and the joining region of T gamma gene. Rearranged bands of T beta gene on one or both allelic chromosomes were detected in all neoplastic T cells, even those of smoldering ATL, in which only a small percentage of peripheral blood T cells were detected as leukemic. T gamma gene was rearranged in the cells of all but one patient, the exception being one ATL patient. In order to test whether any given variable region (V) of T beta gene was expressed in ATL cells, two functionally rearranged V beta sequences of ATL were compared with a V beta sequence from T cells acute lymphoblastic leukemia cells. No significant homologies were noted among the three deduced gene product amino acid sequences, confirming that T beta molecules of ATL cells contained no specific structures in common. The observed heterogeneity of T beta and T gamma gene rearrangements in ATL cells further supported these findings.

TdT+/nonlymphoid antigen+ acute leukemias: immunologic and karyotypic monitoring during therapy and at relapse suggests the transformation of a bipotential stem cell.

Immunophenotype and karyotype were monitored in 19 adult acute leukemia patients with blast cell populations expressing terminal transferase (TdT) and nonlymphoid antigens either at presentation or at relapse. Three patterns of immunophenotypic course were observed when following the patients through at least one, sometimes two (six patients), or three relapses (one patient). Induction chemotherapy induced predominantly TdT+ leukemias with a minor monoblastic component to become TdT-negative, purely monoblastic without clinical response or change in karyotype in five patients (group 1). In group 2, relapse was associated with the disappearance (four patients) or the appearance of TdT+/nonlymphoid antigen+ features (four patients). In two instances, new nonrandom cytogenetic abnormalities, in one case, evolution of an initial abnormal cytogenetic clone, were found at relapse. Six patients (group 3) presented and relapsed with identical TdT+ myeloblastic, promyeloblastic, monoblastic immunophenotype and karyotype. In general, FAB classification did not reflect expression of TdT in nonlymphocytic leukemias or the presence of nonlymphoid blast features in lymphocytic leukemias. Lymphoid-specific antigens in addition to TdT were not detected in any of the cases at the time of nonlymphoid antigen expression. In 11 of the 19 patients, simultaneous expression of TdT and myeloid or monocytic antigens could be demonstrated at the single cell level using double-fluorescence staining. These follow-up data are best consistent with a drug-induced maturation drive of a TdT+/monocytic (majority of cases) or TdT+/myelocytic leukemic stem cell with its differentiation commitment being influenced by chemotherapy or by other as yet undefined conditions predisposing to proliferation of the leukemic cell at relapse.

Epstein-Barr virus-induced increase in the concanavalin-A receptor density of established EBV-negative lymphoma lines in vitro.

Infection of two human EBV-genome negative lymphoma lines with the EBV-variants P3HR-I and B95-8 converts the lines to permanent carriers of EBV-DNA and the EBV-determined nuclear antigen. This process is accompanied by a series of biologic changes similar to those in oncogenic transformation by known tumor viruses (e.g. polyoma, SV40, Rous SV). They include altered growth properties and membrane changes, one of which is expressed in an increased agglutinability by Con-A. Although most transformed cell systems are more agglutinable than the non-transformed counterparts it is controversial whether they possess similar or changed numbers of lectin receptors. Since little was known about lectin receptor densities of virus transformed lymphoma cells, we set out to determine the densities of Con-A receptors on the EBV-negative lymphoma lines BJAB and Ramos and their EBV-converted sublines by simultaneous recording of the cell membrane fluorescence of FITC-Con-A and the cell volume using the FLUVO-METRICELL flow cytometer. We found that EBV infection gave rise to cell lines with a significantly elevated Con-A receptor density compared to the EBV-negative parental lines. The similar changes found in a number of independently converted sublines suggest that they are due to the direct or indirect action of the viral genome.

Variation amongst K562 cell cultures.

K562 cell cultures were obtained from three laboratories (A, B and C) outside our institution, and were designated according to source as K562A, K562B or K562C. The cultures obtained were constitutive or "wild type" K562 cell cultures, not cloned sublines. These cell cultures were compared with respect to growth kinetics, cell surface protein markers, surface antigens, cytogenetics and hemoglobin production. Morphology, growth kinetics in liquid suspension culture, cloning efficiency in soft agar culture, binding of anti-K562 monoclonal antibodies, and the majority of cell surface proteins were generally similar. In contrast, several important differences were observed: (1) hemoglobin synthesis induced by hemin was significantly different among K562A, B and C, K562A being most sensitive (P less than 0.05); (2) whereas more than 90% of K562A or C cells appeared to be Philadelphia chromosome (Ph1)-positive, less than 15% of K562B cells contained a Ph1; (3) membrane proteins (93 and 85 kilodalton) were identified in K562A, whereas only the 93 kilodalton protein was detected in K562B and neither of the proteins were detected in K562C. Our results indicate that K562 cells maintained in different laboratories can undergo tangible changes which may influence experimental results obtained in studies using these cells.