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Characterization of the Continuous, Differentiatjag Myeloid Cell 'Line (HL-60) From a Patient With Acute Prontyelocytic Leukemia ,. . .. By R. Gallayher, S. Collins, J. T r u j i k , ICi.M$redie, M. Ahearn, S.Tsai, -.*.R. Merzgar, G. Aulakh, R. Ting, F. fh'scetti. and R. Gay0 .'% In a preliminary communication," w e described the establishment of a continuous human myeloid cell line (tlL-60). Here we report the detailed properties of this \and produce s u b c u t h OU8 myeloid tumors (chloromas) in nude m A source of colo- ny-stimulating activity 8t ulated the clon- rTcell line and document its derivation fro the peripheral blood leukocytes of a patie t w i t h acute promyelocytic leukemia. A? Characterized by light and electron micrdscopy. the predominant cell type in both t o chemical induction of dlffo esseptially unchanged through the fresh and cultured sources is a neutrophilic promyelocyte w i t h prominent nu- clear/cytoplasmic asynchrony. Up t o 10% of the cultured cells spontanuously differ- chromosomes became predominant w i t h entiate beyond the promyelocyte stage, continued passaging. The most consistent and the proportion of terminally differen- karyotypic abnormalities w e r e the deletion, tiated cells is markedly enhanced by of chromosomes 5. 8. and X and the addi- compounds known to stimulate differentia- tion of a marker resembling a D-group tion of mouse [Friend) erythroleukemia acrocentric and o f a submetacentric mark- cells.'' The HL-60 cells lack specific mark- er. most likely an abnormal E-group chro- ers for lymphoid cells, b u t express surface mosome. No DNA herpesvirus or RNA receptors f o r Fc fragment and complement retrovirus was isolated in the fresh o r (C31, which have been assdciated w i t h cultured cells. The HL-60 cultured cell line differentiated granulocytes. They exhibit provides a continuous source of human phagocytic activity arid responsiveness t o a cells for studying the molecular events of chemotactic stimulus commensurate with myeloid differentiation and the effects of the proportion of niature cells. As charac- physiologic, pharmacologic, and virologic teristic of transformed cells. the HL-60 elements on this process. cells f o r m colonies in seniisolid medium IN MICE, the availability of sustained cultures of erythroid' and myeloid2*'cells has fostered experiments to determine regulatory mechanisms involved in cell replication and dillcrcntiation and to study external factors that may affect these processes.'.' In man, i n vitrti culturing of myeloid cells has primarily been restricted From the Laborutury Id 'i'unwr Cell Biology. National Cancer Institute, Berhesda, Md.; the Departments of Dewlopineniul Therapeutics and Loboratory Medicine. M. D. Anderson Hospital. Housion. Texas; Biotech Keseawh Laboratories. Rockville. Md; the Department of Microbiology and Immunology. Duke University. Durham. N . C.; and the Division of Virology. Bureau of Biologics. Buthesda. Md. Supported in parr by Nurionul Cuncer Institute Contracts NO/-CM-93719 and NOI-CM-02271 and N I H Grant CA-12687. Submitted January 23. 1979: uccrpted May 25. 1979. Address reprint rrqur.yts to Xohrrt C. Gallo. M.D.. National Institutes of Health. 9000 Rockville Pike. Building 37, Rooin 6BOJ. Ueihesda. Md. 20014. 63 I979 by Grunr Ji S~ruttcin1, nr. 0006-4971/79/5403-0018$02.00/0 714 GALLAGHER ET AL. to growth of small colonies for short periods of time in semisolid riiedium,6 which imposes severe experimental limitations. We previously reported ihat human leukemic myeloid cells can be carried in an actively replicating and difl'crentiating state for periods up to several months when cultured in the prcscncc of' conditioned medium from certain human embryo However, there liavc been dificulties obtaining adequate quantities of active conditioned medium for extcnsive studies.' I n one case, after initiation with conditioned medium from a human embryonic lung culture, the leukerliic myeloid cells continued to grow arid dili'crciitiate i n the absence of any-eTogenous growth stimulator. The basic characteristics of this cell line (HL-60);agch&ability to respond to pharmacologic inducers of dilrcrentiation have been re@,rgd, Here we report more detailed information regarding the 'HL-60 line and the patient with acute promyclocytic leukemia from whom it was derived. Recently, Koemer and Golde" also estnblislicd :I diwercntiat- ing myeloid ccll line from a paticnt with erythroleukemia. Prior to llicse reports, there had been numerous successes i n establishing human 1yiiiphobl;ist cell lines, mostly B cell lines engendered by Epstein Barr virus (EBV)." I-Iowcvcr. there had been only two successes with the extended propagation of huinan niyeloid cells, and i n neither case was differentiation observed i n suspension culture."." Recently, Karpas et a1.I6 have also reported the establishment of huriiart leukocyte cell lines with a few myeloid features, but these lines primarily have the properties of B-lymphoblast cells, including the presence of EBV and s u r l k c iriiiiitiiioglobulins. MATERIALS AND METHODS Case Report S.G.. a 36-yr-old white female. was referred to the M . D. Anderson Ilospital aiitl Tuiiior Institute with a diagnosis of acute promyelocytic leukemia. She had been well until 2 1110 previously when following an upper respiratory infection. she developed easy bruisability. swollen giinis, and bone p i n . On the admission physical examination. she had multiple small nodules, I crii or less in diameter, over her scalp, cervical lymphadenopathy. and a 6 x 8 cm nodule in the left lateral vaginal w:iII. Numerous ecchymotic spots and petechiae were present over the arms, trunk. and lcgs. The liver cxtcnded I O cm below the right costal margin. the spleen 3 cm below the left costal margin. Afliiiissioii peripheral blood counts were as follows: hemoglobin 9.3 g/dl. hematocrit 27.4%. platelets XO.OOO/cti iiiiii.white blood cells 35.700/cu mm with 70% myeloblasts and promyelocytes, 14% myelocytes, 2'3, hands. and 14% lymphocytes. The bone marrow showcd 4.06 blasts. 85% promyelocytes. 2.8%' myclocytes. 0.8% metamyelocytes. and 1% normoblasts. The cells were heavily granulated. and special \tnins showed peroxidase positivity and PAS negativity. The Serum lactic dehydrogenasc ( L I ) l 1) activity was elevated to 650 r U / m l (normal 30-1I O U):other values of the S M A I2 autoanaly7er were within normal limits. Coagulation parameters. including prothrombin time, partial thromboplastin tiiiic, lihrinogen, and fibrin split products were within normal limits. Treatment was initiated with intr;ivcnoiis C-parvum, systemic prophylactic antibiotics, and chemotherapy with adriamycin. vincristine. prednisone, and cytosine arabinoside. There was a partial response to two initial coursesor i l i i s thcr;ipy. \rill1 a reduction in the WBC to less than 2000/cu mm and a decrease i n the marrow leukcriiic infiltr;itioii and in size of the subcutaneous nodules. However, disease progression was noted during ii third course 0 1 chemothera- py. On the 64th hospital d a g the patient complained o f the sudden onset of nhdoiiiiiial pain and succumbed to intraabdominal hemorrhage and shock. The coagulation prolile. which up t u lhis time had been normal. showed evidence o f a consumptive coagulopathy with iiinrkcd prolonpition of the prothrombin time, an increase in the partial thromboplastin time. and a rcduction (if the serum fibrinogen level to 36 mg/100 ml. At autopsy, there was evidence of leukemic inliltrates i n the mediastinum. lymph nodes, uterus, vagina, and ovaries. and there was it iiiasivc hcriiriperitoncum, splenomegaly, and multiple petechial hemorrhages. The source of bleeding W:IS ihc splccii. which had a subcapsular infarct and capsular tears. HI E bl dc P' 'P ni th c ch m' Pa Ff trt mg lei 0.1 tel su (P E1 1.2 Wil let EP RO Cf l els' Per Ig/ ant al.! an! Wa. spe elel C/i ( Inc bob st. 13dial huii HL-60 IN ACUTE PAOMYELOCYTIC LEUKEMIA 715 Establishment vj'the Prvtnyelocytic HL-60 Cell Line in Suspension Culture Prior to treatment of the patient, peripheral blood leukocytes were obtained by leukopheresis. using a blood cell separator. `I'liecells were shipped on wet ice to the NCI and on arrival were 98% viable. as determined by trypui blue dye exclusion. As previously detailed.'" the leukocytes were cultured in the presence and absence uf conditioncd medium from human embryonic lung cultures. After a 3-wk lag period, active gruwili of cell5 in auspension was observed in flasks supplemented with conditioned medium from one hiiiiiaii embryonic lung culture. Subsequently, it was found that continued growth of the leukocytes did nut require conditioned medium supplements. Cytogenrrir Stir(1ir.s ' Cytogenetic exaniinations were done on the following samples: ( I ) fresh bone marrow prior to chemotherapy;(2) cultured 11L-60cells frompassages6.8. I5,28.35.45.and55.grownonRPMI 1640 medium with 10% Iicul-inactivated fetal calf serum, or from passages 21 and 41,grown for the last 6 passages on Ham's 1:- 10 inrdiuin with 20% fetal calf serum; and (3) after passage through nude mice. For analyses, the cells were incubated in the presence of colcemid. 0.02 pglml. for 1 hr at 37*C, then treated with 0.075 bl KCI for 20-30 min at room temperature. and fixed in three changes of methanol-acetic acid (3: I ). Chromosome preparations were made using air-drying or flame-drying techniques. Chroiiiomiiics were banded by the trypsin-Giemsa method; slides were treated with 0.01%-0.03% trypsiii i n phoq)hate-buRered saline (Ca'+ and Mg'+ free) for 3-6 min a t room temperature and stiiined with 4%Giemsa stain in Sorenson's butrer at ptt 6.8. Histochemical S'toitiitigPrut.rdurrs Published procedures were fullowed for Napthol ASD chloroacelale and u-methyl acetate esterases." Sudan black B.I9 ii1yel~ipcroxiJesca.~lkaline and acid phosphatase^.'^^" and the periodic acid SchiR (PAS) reaction." Electron Microscvpic Yrcymration The initial bonc niiirrow and sibsequent cultured tIL-60 cells were mixed in 2.5% gluteraldehyde. pH 7.2, in Sorenson's plio*pli;rh: butler, 300 mosmol, for I hr at 25C. Dianiinobenzidine tetrahydrochloride was utilized to dciiiarwte endogenous peroxidase activity. The tissue was post-fixed in 1% osmium tetroxide, p t l 7.2, fur I lir. Following dehydration in graded acetones, the cell pellets were embedded in Epon 812. Section5 *ere CUI oil an LKB ultramicrotome and examined with a Siemens Elmiskop 102 at 80 kV. Cell Surface SIiidirs Erythrocyte roxtte tebts were performed by slight modifications of established techniques, as detailed elsewhere.'' The I:-rorctte assay utilized sheep erythrocytes. The EA and EAC-rosette tests were performed with both slicep iiiiil h v i n e erythrocytes. Tents for surface immunoglobins IgG. IgM. and IgA utilized immuri~ibcads(Uiu-Rad Laboratories). as detailed elsewhere." Direct testing of HLA antigens was done d l IhAe University by the standard two-stage microcytotoxicity method of Amos et al." and by the iiictlid or Miclal et al." with a panel of sera used to define HLA-A. E. and C locus antigens at past l i b \ lli~tocoiiipa~abilitTyesting Workshops. Direct testing for human la-like antigens was done by conililcincnt dependent microcytotoxicity*' using rabbit and monkey antisera known to specifically detecl IJantigms by cytotoxicity and by radioimmunoprecipitalion and polyacrylamide gel electrophoresis analysir.'* Chemotaxis S I rrilirs Cells were added L ~ Ithe upper well of a blind well chamber (model F013 WLB 00101 Neuro Probes, Inc.. Bethesda. M d . ) at a cuiiccntration of 2.5 x IO6 nil of Gey's balanced salt solution containing 2% bovine serum albumin kind 0.01 Af Herpesn The dipeptide N-formylmethionyl-leucine(Sigma Corp., St. Louis, Mo.), a putent chcnioattractant~'was added to the lower well at a concentration of IO-'M. A I3-mni chemotactic nicmbraiie (Nucleopore Corp., Pleasanton, Ca.), containing an average pore diameter of 5.0 p , ,elxirated the LWO compartments. The chamber was then incubated at 37'C in a humidified 5% CO, atniusplie~efor 1 hr. The filters were then removed, stained with WrighbGiemsa. .. 716 GALLAGHER ET AL. and examined microscopically for cells that had migrated IOthe underside. Results were expressed as the number of cells counted in four successive low power fields. All experiments were pcrfornied in triplicate and the results averaged. Phagocytosis Studies Cells were suspended at Id cells/ml in RPMl 1640 medium supplemented with 10% FCS. Cundida ulbicans. grown overnight in Sabouraud's medium (Difco, Detroit, Mich.). were washed twice with saline and added to the above I4L-60 cell suspension at 4 x Id organisms/ml. The suspcnsicin was then incubated for 30 min at 37OC. and the percent of cells that had phagocytosed yeast particles was determined by light microscopy on Wright-Giemsa stained slides. Lysozyme Studies Cells. at a concentration of IO'/ml. were lyscd by incubating with 1% NP-40 i n PDS. pi I - 6.2. for IO * min at room temperature. The cell lysate was then spun at 3900 rpm at 4 O C for 10 win, and the lysozyme assay was performed on I ml of the supernate with egg white lysozyme (Sigma) used as the standard." I n addition, lysozyme assays were performed on I-ml aliquot samples of the cell-free supernate of actively proliferating cells harvested at a time when cells were at a concentration of 1.5 x 106/ml. Growth and Cloning in Semisolid Medium To assay for colony formation. various concentrations of HL-60 cells were suspendad in 1.2% methylcellulose (Dow Chemical Corp.) or 0.3% agar (Difco) in McCoy's 5A niediuni containing 10% fetal calf serum (FCS) and 5% horse serum. One milliliter of this mixture was plated in 35-nim culture dishes (Falcon) and incubated in a humidified 5% CO,chamber at 37%. Colony counls (230cells) were performed after 7, 14. and 21 days of incubation. For cloning studies. the IIL-60 cells were suspended at a concentration of 10zcells/ml in 1.2% methylcellulose in McCoy's SA medium containing 10% FCS and cultured as above. After 5-6 days, visible colonies containing 20-30 cells were individually transferred to wells of a 16-mm multiwell dish (Linbro Chemical Co.. New I Inven. Conn.) containing 0.5 ml of RPMl 1640 medium supplemented with 10% FCS. The cells wcrc then recloned using the same procedure. As cell growth continued, cells were transferred to larger wells and ultimately to Falcon T / 6 0 flasks. Growth in Nude Mice Athymic nude mice (BALR/c nu/nu) were obtained rrom ARS/Sprague Ihwley (hlatliron, Wisc.). HL-60 cells at dimerent passage numbers were injected subcutaneously into the mice. Tumors that developed were dissected, stained with Wright-Gicnisa and myeloperoxidase, :and an;~ly;.cdfor karyotype. A separate cell line (designated HL-60 Nu,) was established by mincing a tumor obtained from injection of HL-60 at passage 45. incubating the tumor mince with 0.25% trypsin for 30 niin at 3 7 T , resuspending the resultant cell suspension in RMPI 1640 plus 15% heat-inactivated FCS, and continuing the leukocyte suspension cultures under standard conditions. Testfor Herpesviruses The Epstein-Barr virus nuclear antigen (EBNA) test was performed by R nicu1ilic:ition of the Reedman-Klein technique.*'.n Herpessimplex type I and cytomegalovirus (Town strain) probes were prcparcd by Iabcliirg the DNA with tritium in vitro by "nick translation," using all four 'H-labeled nuclctside triphosphates to a specific activity of 1.5 x IO' cp,m/pg as detailed by Maniatus et aLm These probes were hybridized to excess cellular DNA up to a fi`nal Ecot of 8000, and the hybrids were processed by hydroxyapatite chromatography.'' TestsJor Retroviruses To test for possible extracellular virus, up to 100-ml samples of H1.-60 cell cultiire fluid were concentrated and assayed for RNA-directed DNA polymerase (reverse tr;inscriptase) activity, as previously reported." In addition, cultures were tested for release of R NA-containing particles by HL-U pulse ultrac At1 5'-icd testcc Th exam searc mic n by set retro! detec weigl adenl retrot indirt acids Final with Gro A pres cult1 leuk vi 3.0 pass sho\ pres wit1 Fi on 11 and perfr and COUl the i of c cult1 half werf RPM 50 b fetal A.A. seru wen seru d r AL. IS the icate idida with then War )r IO I the I the .free .5 x I .21 10% Lure ells) #ere ning mere nn.) mcd llely sc.). that ryotom IT. a nd the NA oa 1 to tite ere BS by HL-60 IN ACUTE PROMYELOCYTIC LEUKEMIA 7 17 pulse-labeling up to 50-1111culture samples with 'H-uridine. concentrating the labeled particles by ultracentrifuuyaiion, and centrifuging them to equilibrium on a 20%-60!% sucrose gradient." Attempts tu induce virus production followed published procedures. including treatments with 5'-iMlodeoxy11ridi11c,'`dimethylsulfoxide.'0 and arginine-deprived medium." 5-Iododeoxyuridine was tested with and without the addition of hydrocortisone, IO-*M. The possible presence of subvirion components within fresh and + u r d HL-60 leukocytes was examined by a variety of tests. From 2 to 25 g of viable, nonfrozen cells were used per test. Methods to search for iiitracrllular reverse transcriptase. included the preparation and purification of the cytoplasmic microsoiiial-iiiembranc fraction'* and detergent extraction of whole cytoplasmic extracts followed by sequential column chrumatography." Tests for cellular antigens related to p30 proteins from known retroviruses were pcrfuriiied on whole cell extracts using a competition radioimmunoassay capable of detecting I ng of cumpcting virus-related antigcn." Attempts were made to demonstrate high molecular weight RNA associaled with intracytoplasmic virus-like particles by direct pulse-labeling with 'Hadenosine.M Also, cytoplasinic RNA was tested for possible relatedness to RNA from known retroviruses by IW mclliods: ( I ) direct hybridization assays with 'H-cDNA viral probes" and (2) indirect compclitiun assays in which cytoplasmic RNA is tested for possible relatedness to viral nucleic acids by its ability to inicrfcre with the hybridization of homologous viral R N A to proviral DNA.'' Finally, the H 1.-60 cellular DNA was tested for viral-related proviral sequences by direct hybridization with tritium-labclsd type-C viral cDNAs" and with "'I-labeled whole viral RNAs." RESULTS Growth Kinetics in Suspension Culture As previously reported," the HL-60 blood leukocytes initially grew only in the presence of culture h i d supplemented with conditioned medium harvested from cultured human embryonic lung cells. However, after a few weeks, the cultured leukocytes grew as well in the absence as in the presence of conditioned medium supplements. At passage 9, the saturation density of the cultured cells was 2.33.0 x IO6 cells/ml, and the cell-doubling time was 55-60 hr." With continued passage, llic tlL-60 cells have continued to adapt to in vitro culture conditions. As shown in Fig, I , the growth of the cells has remained absolutely dependent on the presence 01' I-CS through 68 passages. Maximum growth stimulation was achieved with 10%-2~0% FCS. t Iowever, at a given serum concentration, the saturation Fig. 1. Effect of serum concentration on the growth of HL-60 leukocytes at low and high passage levels. Cell counts were performed at passage 17 (closed symbols) and passage 68 (open symbdsl. using a Coulter counter. Plotted points represent the average OF triplicate counts from eech of duplicate 10-nil culture ftasks. The cultures were refed on day 6 by replacing half of the culture fluid volume. Cultures were carried. as previously detailed." in RPMI-1640 mediuni containing gentamicin, 50 Irg/ml, and varying concentrations of fetal calf serum as follows: 6.0. no serum; &A, 1% seruiii: 1.0 6% serum: v.v 10% serum. Curves observed using 20% serum were superiniposible on those with 10% serum. 70- a- a o 2 4 6 8 1 0 1 2 1 4l DAYS IN CULTURE HL-60 IN A density h level. At cel I-doub these vali was also the cultui Cytologic The ce adhere to occasiona there is cc larger cel Figure: Giemsa-si cultured prom ycloc distinct n basophilic intensive specimens including Sudan bla tasc, :I st: promyeloc acid-SchiI The inorc negative f esterase w ceI Is." As sum1 tiate into banded an' size, decre tation. dec Passags Numba Fresh. ot 9 18 35 63 *From diffe tFrom difk ?I ,?d ir: 4L '1 >. p b h. & s from iavert. rior t o Cytoqtes. e ASD Is. (GI owing eosin HC-60 ssent. Nith a HL-60 IN ACUTE PROMYELOCYTIC LEUKEMIA 719 density has increased and the doubling time has decreased as a function of passage level. At passage 17, the saturation density was 5.5 x IO6 cells/ml, with a cell-doubling tiinc ol' 36 hr during the logarithmic phase of growth; at passage 68, these values were, respectively, 7.6 x IO6 cells/ml and 30 hr. Continued passage was also accompanied by some shift i n the cytologic a'nd karyotypic properties of the cultured cells (see below). Cytologic C'hamctrristics of Fresh and Culrured Cells The cclls grow in single-cell suspension without any tendency to clump or to adhere LO plastic or glass (Fig. 2A). The cells are generally round or ovoid, and occasional cclls have blunt pseudopods. The modal cell diameter is 13p; however, there is considerable variation in cell size, ranging from 9p to 2Sp in diameter. The larger ccllr are I'ruqucntly binucleate. Figures 2U arid 2C illustrate typical leukemic myeloid cells observed in WrightGiemsa-htaincd prqxirations from the patient's fresh blood specimen and from the cultured 111.-60 cells. In both instances, the predominant cell is an abnormal promyelocyte. This cell typically contains a large round nucleus with regular, distinct riiargins, fine chromatin, and 2-4 nucleoli. The cytoplasm is deeply basophilic arid contains multiple prominent azurophilic granules. Despite an intensive search, Auer rods were not observed in either the fresh or cultured specimens. These cells stained heavily with stains specific for myeloid cells, including myelopcroxidase (Fig. 2D), ASD chloroacetate esterase (Fig. 2E), and Sudan black B (Fig. 2F). However, no positivity was noted with alkaline phosphatase, a shin characteristically positive in normal neutrophilic granulocytes. The promyelocytcs sliowed slight diffuse staining of the cytoplasm with the periodic acid-Schill reagent, and occasional cells stained positively for acid phosphatase. The more mature granulocytes stained strongly positive with PAS and were negative for acid phosphatase. Stains for a-naphtol AS-D acetate (nonspecific) esterase wcrc iiniI'ornily negative. This staining pattern is characteristic of myeloid cei Is." As summarized in Table 1, 5%-10% of the myeloid cells spontaneously differentiate into inore miiture granulocytes, including myelocytes, metamyelocytes, and banded and segmented neutrophils. This process is accompanied by diminished cell size, decrciiscd nuclear-cytoplasmic ratio, increased nuclear pycnosis and segmentation, decreased cytoplasmic basophilia, and replacement of the coarse azurophilic Table 1. Differentiation of HL-BO Cells as a Function of Culture Parrage ~~~ Percent of MyelcudCella Passage Number Myalobldsts to Prornyelocytss Myehytsr Metamyekcytes to .Segmented Nwtrophib Pawnt Inbcibb by 1.25% DMSO Fresh' ot 9 18 35 63 81 95 89 92 97 96 16 4 7 4 1 2 Not tested Not tested 70-90 70-90 70-90 70-90 *From differentialWBC 011 fresh blood specimen at M.D.Anderson Hospital. tFrom diffurentialWBC on leukopheresisspecimen prior to culture at NCI. EA ET AL. HL-60 IN ACUTE PROMYELOCMIC LEUKEMIA 721 c . 1 .. :* Y b., romyelo,logy and n in vivo. leer bleb ncy with oplasmic {toplasm specific 11 in the are also erved in the inltial bone marrow specimen of this patient. The developlng nuclai are prlnurily bla8tic in appearence. whereas t h e cytoplasmic areas ere prornyelocytic (xW00). ( 0 )This "band" or "stab" HL-80 cell displays evidence of nuclear and cytoplasmic differentiation. Although the granules are malformed. both peroxidase-positive azurophilic (large arrow) and peroxidase-nogative specific (smallarrow) forms are presant 1x2O.OOO1. (El Following dimethyl formamide (60mM) incubation for 8 days. the nuclei of HL-BO display dramatic nuclear segmentation and increased definition of heterochromatic areas. Cytoplasmic granules are of both the azurophilic (large arrow) and specific (smell arrow) type ( x 39.0001. I 7 2 2 GALLAGHER ET AL. granules by smaller specific granules. As reported in detail elscwhcre," the proportion of differentiating promyelocytes could be greatly cnhanccd by adding certain compounds known to induce dilferentiation of mouse (Friend) erythroleu- kemia cells, e.g., DMSO,to thecultured HL-60 cells (Fig. 2G).DiKerenliation was limited to the neutrophilic series in suspension culture, sincc no eosinophilic or basophilic granules were detected in HL-60 cells after eithcr spontaneous or induced differentiation. In addition to the neutrophilic grannlocytcs, a minor population of cells resembled monocytes with folded nuclci, palc-st;iining blue cytoplasm, and less distinct and regular cytoplasmic margins. N o cells rcwnbling immature erythroid cells were observed, and benzidine stains for hemoglobin were negative. With continued passaging of the HL-60 cells, the pcrccntagc of myeloid cells showing spontaneous terminal differentiation has diminihlicd slightly. This did not substantially alfect the induction of dimerentiation by DMSO (Tnblc I ) . Electron Microscopy o$ Fresh and Cultured Cells Ultrastructurally, the HL-60 line consists of a heterogeneous population of cells with the predominant morphology varying from myeloblastic to proiiiyelocytic. Nuclear/cytoplasmic asynchrony is present, the cytoplasm rcprcsenting the more mature element. The nuclear chromatin is primarily euchromatic, and wcll developed nucleoli are situated adjacent to the nuclear membrane. Cytoplasmic granulation in the cultured cells is mostly of the azurophilic type. The s i x , shape, and density of these peroxidase-positive granules (Fig. 3A) is morc divcrsc than those observed in the patient's original bone marrow (Fig. 3B). I n this initial specimen, 85% of the cells are characterized by blast-like nuclei and progranulocytic cytoplasm (Fig. 3C). I n addition to the nuclear/cytoplasmic asynchrony, nuclear blcbs and bundles of cytoplasmic fibrils are present i n leukemic cells both in vivo and i n vitro. Under routine culture conditions, a small percentage of these cells deiiionstratc "stab" or "band-like" nuclei and evidence of both azurophilic and specific granulation of the cytoplasm (Fig. 3D). The addition of the differentiating agcnt. dinicthyl formamide, to the HL-60 culture medium induces further segmentation of the nuclei and increased specific granule formation in the cytoplasm (Fig. 3i4,). Cytogenetic Analysis of Fresh Bone Marrow A total of 40 metaphases wcre analyzed from the initial bonc ni;irrow sample. The analysis showed 31 cells with 44 chromosomes (approxiitlately 7570). 8 with 45 (20%) and 1 with 46 (5%) (Table 2). Examination of these clones, with the help of -Giemsa-banding techniques, indicated that the common alterations i n all of the Table 2. Distribution of Chromorome Counts of HL-60 Calls as a Function of Culture Passage PSSS8ge Numb 0 (Fresh) 6 and 8 15 and 28 35 45 55 Metaphases Counted s43 Chranosome Numbrr 44 45 46 4 7 548 -4 0 31 8 1 - - 145 6 87 18 26 6 2 105 2 1 47 5 1 4 50 3 7 29 83 90 2 5 64 89 2 71 2 4 47 78 3 HL-60 II cells st resem metap D-gro karyo, 4 MI 1 -6 c I 1 4 1 P 4* I; t 1 Fit kery saml bone (44.) somi HL-60 IN ACUTE PROMYELOCMIC LEUKEMIA 723 +cells studied wcrc iis follows: - 5, - 8, and a submetacentric marker chromosome resembling an E-group chromosome (M3) (Fig. 4A). Additional changes in one metaphase included the presence of a marker acrocentric chromosome resembling a D-group chroinosome ( M 2) and an extra chromosome 18. The pseudodiploid karyotype shown in Fig. 48 illustrates these additional changes. Other marker illt 5 % 13 4 5 J r 811 6 89 G1 12 Lw 13 a fa 19 A "4 8 1 Cr 21 ,Ir M3 tfH 3 40 18 b'r xx Q 5 btr 11 iElX 6 89 44 12 hcr 13 (10A 18 i wa. - 0 19 21 x x B 4 *+ M2 M3 Fig. 4. Karotype analysis of fre8h. uncultured HL-80 bone marrow 8pecimen. (A) Hypodiploid karyotype with 44 chromosomes. reprssanting the main clone (76%)found in the initial bone marrow sample of the patient 144. XX,-5,-8,-17.+M3). ( 8 )Paeudodiploid karyotype found in tho initial bone marrow sample of the patient 15%) in the in vitro leukemic call linea. passage 28 (60%) (44,XX.-5.-8,-17. t 18,i-MZ. -t M3). The two markers mo8t likely repre8ent a D-group chromo- lome (M2)and a 17pi 1M3). 724 GALLAGHER ET AL. chromosomes, double minute chromosomes, and structural alterations involving otlicr C-group chromosomes (numbers 7,9, IO) were also occasionally observed i n a fcw hypodiploid metaphases. These variant karyotypes differed froin cell to cell and were difficult to categorize even with the use of Giemsa-banding techniques. Cj~togcneticAnalysis of C'itltirrcdIiL-60 Cells During the early passages in vitro, the modal chromosome number was 44.as in the fresh leukemic bone marrow (Table 2). However, with continltcd passaging. the modal chromosome number shifted to a mixed population with 45 or 46 cliromosomes. Using the Giemsa-banding technique, considerable variation wits notcd with respect to individual chromosome composition. However, as in the rresh marrow, the following karyotypic alterations were commonly found: - 5 , - 8 , + :III :ibnor- + +mal E-group chromosome, 18. and an acrocentric D-group markcr. Altliough inconsistent, other alterations includcd missing C-group chromosoliics 9 ;incl 10, loss of chromosome 16, loss of chromosome X, the presence of a variable nunibcr of double minute chromosomes, and translocation of additional chromatin material to chromosome 7. Figure 5 illustrates a frequently observed karyotype with 45 chromosomcs that emerged after passage 35. It includes 7 recognizable abnormalities as follows: ( I ) +-5; (2) -8; (3) -16; (4) - X; ( 5 ) an A-group marker ( M I ) ; (6) + an r! 5 ;': 12 $4 19 8 X M1 M2 H3 Fig. 6. Karotype analysis of tho predominant tissue culture clone of HL-80 cells after passaga 36 {4S:-X.-6.-8.- 16,+Ml.+MZ.+M31. HL-60 IN I acrocent (M3).tf passages bone ma chromos1 Cell Sur The rc ize h um: characte tin, char. the tIL-1 cytes. bu fornii tig bovine er eryt hroc! increasec cultured usually FC Micro1 antisera could be membrar membrar zee antis prepara ti HLA t HLA-A I quantitat some indi Cellular In add HL-60 cc transfer a : E rosette EA rosette EAC rose? Surface im (W.Igr la-like antit *Some 2' lymphocytes ER ET AL. rivolving -ved in a cell and -14, as in ;ing, the chromoted with narrow, 1 abnorlthough and 10, ,mber of terial to lies that ows: ( I ) ) + an li 5 $4 12 61 ia 6 X aasage 36 HL-60 IN ACUTE PROMYELOCYTIC LEUKEMIA 725 +acrocentric D-group marker (M2); and (7) a submetacentric E-group marker (M3). All alterations except no. 5 were also noted commonly in the earlier passages. The MI marker was only rarely observed in metaphases from the fresh bone tiiilrrow. The M3 marker appears to correspond to the abnormal E-group chromosome, but its identity remains to be determine9 with certainty. Cell Surfuce Afnrkers The results of surface marker tests, which have been generally used to characterize hunian leukocyte cell lines, are summarized in Table 3. The E-rosette test, characteristically positive with T lymphocytes, and tests for surface immunoglobulin, characteristically positive with B lymphocytes, were uniformly negative with the HL-60 cells. The EA test for Fc receptors was negative using sheep erythrocytes, b u t was quite positive using bovine erythrocytes, with over 50% of the cells forming roseltcs. Conversely, in the EAC test, no rosetting was observed using bovine erythrocytes, but 2%-9% of the cells formed EAC rosettes using the sheep erythrocyte assay. The percentage of sheep cell EAC-rosette-forming HL-60 cells increased up to sixfold after induction of differentiation by DMSO. Control cultured T lyniphocytes (Molt-4) and undifferentiated myeloid cells (K-562) usually formed no EAC rosettes using sheep erythrocytes. Microcytotoxicity testing of HL-60 cells with high-titered rabbit and monkey antisera to human la-like antigens was negative. Moreover, no Ia-like antigens could be immunoprecipitated from 'ZJI-labeled,deoxycholate-solubilized HL-60 membrancs with these antisera. More detailed studies on the nature of other membrane antigens of HL-60 cells and the characterization of rabbit and chimpanzee antisera to the cell line will be published separately (Metzgar et al., in preparation). HLA typing revealed that HLA-Bwl7 was a major marker at all passage levels. HLA-A 1 (predominant) and HLA-A2 were also detectable but varied somewhat quantitatively at different passage levels. In addition, there were reactions with some individual typing sera defining other minor HLA-A, B and C antigens. Ceflulirr EnzynwJ In addition to enzyme assays by histochemical staining procedures, extracts of HL-60 cells wcre tested for lysozyme (muraniidase), terminal deoxynucleotidyl transfernsc, and reverse transcriptase. Table 3. Surface Marker Studies Performed With Cultured HL-60 Leukocytes HL-BO Cell Test RaSUlt. flewted L d l o c ~ t M ~ At umocbtian E rosette EA rosutte EAC rosette Surface immunoglobulins (IgG. IgM. 18.41 la-like antigen _. + ** - - T lymphocytes" Fc receptor lymphocytesand diffaentiated granu~ocytes"'~~' Complement IC31receptor: B lymphocyte^.^^ differentiatad granulocytes" B lymphocytesu B lymphocyte^.^' immetwe gran~locytes~.~' *Some 2%-9% of HL-60 cells formed EAC rosettes with sheep cells versus 0%-2% of control T lymphocyles (Moll-41 OT undilferentiatedmyeloidcells (K562l. 726 GALLAGHER ET Al. Lysozyme, an enzyme marker for leukocytes of the neutrophilic-nionocylic cell was detected at a concentration of 4.7 pg/10' HL-60 cells. It was also detected at a concentration of 2.3 pg/ml in the cell culture fluid after growth of HL-60 cells to a density of 1.5 x IO6 cells/ml. N o intra- or extracellular lysozyme was found in a myeloid blast cell line (K562) or in a B-lymphoblast cell line (NC37). The values obtained with the HL-60 cultured cells are simihr to those reported for a differentiating mouse myelogenous leukemic cell line44and for fresh and cultured myelogenous leukemia cells:48they are substantially less than values reported for fresh or cultured leukemic leukocytes with prominent nionocytic Terminal deoxynucleotidyl transferase is an enzyme that may be specilic for immature T lymphocytes but which has been detected in soiiie cases of human ''myelogenous leukemia lacking a prominent lymphocyte cell pcipulation.5" This enzyme was not detectable in extracts of fresh or cultured HL-60 cells, Le., incorporation of 'H-dGMP was less than 0.1 nmole/hr/109 cells using a primer of oligo (dA)12.w Reverse transcriptase, an enzyme specific for RNA retroviruses. hiis been partially purified from the blood leukocytes of a few patients with acute rnyelogenous leukemia" or, in one case, from the spleen of a patient with myelofibrosis prior to conversion to acute myelogenous le~kernia.'T~o date, this enzyme has not been identified in any human culture cell line. I n the present studies, up to I O g of fresh and cultured HL-60 cells were extracted and analyzed for reverse transcriptase-like activity. A cytoplasmic DNA polymerase activity was identified that transcribed a synthetic RNA template better than a synthetic DNA template, Le.. incorporation of 'H-TMP was 152 pmole/hr/lO' cells with oligo (dT)12-18.poly(Aa)nd was 43 pmole/hr/ IO9 cells with oligo (dT)12.,8.p~Iy(dAH).owever, this polymerase failed to transcribe more specific primer templates for RNA-directed D N A polyuierase, oiigo(dG),,-,,.poly(C) or ~ligo(dG)~~_,~.poly(MaenCd )i,t was not at all inhibited by antisera that inhibit (>90%) reverse transcriptase from thc woolly monkey sarcoma-gibbon ape leukemia virus group or from the baboon cndopcnous virus group. This pattern of results is most consistent with h u m a n cellular DNA polymerase y activity rather than reverse tran~criptase.~'"~'~ Cell Funcfional Studies Cultured HL-60 cells were tested for their ability to phagocytize yeast particles and to respond to a chemotactic stimulus, as described in Materials and Methods. On repeated testing, 5%-10% of the cultured cells were capable of ingesting one or more particles of Candida albicans. This corresponds to the percentage of terminally differentiated granulocytes in the culture (Table I ) . As presented in Table ?, the ability of the cultured HL-60 cells to migrate through a 5p pore size mekbrane was stimulated about fourfold by the powerful chemoattractant dipeptide N-formylmethionyl-leucine.In the absence of chemoattractant, the number of migrating HL-60 cells was the same as cultured leukocytes that fail to respond to chemotactic stimuli. This corresponds to the anticipated properties of HL-60 cells as partially differentiated neutrophils visd-vis K-562 cells (undifferentiated myeloid cells), NC37 cells (B lymphoblasts). and Molt-4 cell (T lymphoblasts). M-60 IN A( C ti' K N N *4LPF. 4 tNumbe, Both I en hancel ties of t presente Growth The t passage efficienc sources 5-3O-fol In addit clones, I have no counts c unclone, Tumor The r HL-60 c no inst: distant the turr which v tion of I one suc (3/3) : Un fort I *3-5 tNu,. of passag .R ET AL. /tic cell /as also lwth of sozyme ell line Q those )r fresh values nocytic ific for human 52 This Is, Le., m e r of s been yelogeis prior i t been )f fresh tse-like ribed a xation was 43 : failed nerase, hibited ionkey s virus DNA Irticles :thods. one or termi- iigrate werful :moatocytcs ipated K-562 dolt-4 HL-60 IN ACUTE PROMYELOCYTIC LEUKEMIA 727 Table 4. Chemotaxis Assays of Cultured Leukocytes con Culitue HL-60 K 562 NC-37 Moll-4 MiwtingCdl./4LPF* - Qlsmosttractant 3 * 2t 0 5*2 0 L + Ctwmoattrsctant 17 i 7 2+1 322 0 *4LPF. 4 low-power microscope fields (160x1. tNumbers equal the average number of migratingcells i the observed range from triplicateexperiments. Both thc phagocytic and chemotactic functions of the HL-60 cells were greatly enhanced by induction of further differentiation with DMSO.The detailed proper- ties of thc induced 1lL-60 in comparison to normal mature neutrophils will be presented in anothcr report (S. Collins et al. in preparation). Growth otrd Clotritrg in Semisolid Medium The HL-60 cell> formed colonies in both methylcellulose and agar. At culture passage 16, the plating efficiency was 0.5% * 0.3%, while at passage 85. the plating efficiency was 8.0% 2 3.2%. When the HL-60 cells were stimulated with various sources or colony-stiinulating activity (CSA), the plating efficiency was increased 5-30-fold, as will be presented in detail elsewhere (Ruscetti et at., in preparation). In addition, over I50 clones of HL-60 cells, presumably representing single-cell clones, have been picked and propagated from methylcellulose. Although these have not as yet been analyzed in detail, only minor variations in differential cell counts of culturcs derived from the clones have been observed in comparison to the uncloned culture. Tumor Forttrcrtioti it1 Nude Mice The number of mice developing tumors after injection with different passages of HL-60 cells is shown in Table 5.All tumors developed at the site of injection and in no instance did postmortem examination show any evidence of metastases to a distant sitc. klacrouxpically, all the tumors had a greenish hue. Microscopically, the tumor coiisisted predominantly of prornyelocytes and myeloblasts (Fig. 2H), which were strongly peroxidase positive. Karyotypic markers confirmed the derivation of the tumor from HL-60 cells. The cell culture HL-60 Nu, established from one such tumor was most tumorigenic, with all mice injected developing tumors (3/3) and with as few as lo' cells required to produce tumors (Table 5 ) . Unfortunatcly, i t hiis not been possible so far to serially transplant the nude mouse Table 5. Formation of Tumors by HL-BO Cells in Athymic Nude Mice Fresh Passage 15 Passaye 45 Nut Not done 1/a 318 313 *3- 5 x 10' cells inoculated subcutaneously. tNu,, suspension culture derived from an explant of a subcutaneous tumor originally formed by inoculation of passage 15 tlL-60 cells. , 728 GALLAGHER ET AL. Table 6. Tests of Cultured HL-60 Cell DNA for Herpe8virur-Related Nucleic Acid Sequences Soucs d UnlabeledDNA DNA Pr& Hybrichzsd 1%) HSV-1 CMVt HL-60 Uninfected human cells Infected human Cells 4.0 3.9 85.0 29 2.6 76 3 `Herpes simplex vwus type I (courtesy of Dr. Alvaro Pugal. tCytomegalovwus (Town strain). tumors for more than three generations either by direct in vivo passaging or by reestablishment of the HL-60 Nu, cells in tissue culture betwecn inoculations. Also, no success has been encountered i n attempts to develop an ascites tumor by intraperitoneal injection. The HL-60 Nu, culture has not bccn productively infected by a mouse retrovirus, as determined by reverse transcriptnsc assays or electron microscopic observation. Testsfor DNA Herpesvirus Information The HL-60 cultured cells were repeatedly demonstrakd to be negative for Epstein-Barr virus (EBV) by the nuclear antigen (EBNA) test. This assay has been reported to be 100% effective in detecting intracellular EBV DNA." Nucleic acid hybridization analyses were performed to test for two other herpcsviruses that have been associated with transformation of human cells, cytomegalovirus, and herpes simplex virus. Tests for both viruses were negative at a sensitivity capable of detecting less than 0.1 virus genome copy per cell (Table 6). Testsfor R N A Retrovirus Information Extensive tests were performed on the fresh and cultured IIL-60 leukocytes in attempts to detect retrovirus information according to proccdures described or referenced in Materials and Methods. No evidence for the relcase of extracellular virus-like particles was observed from the cultured leukocytes either spontaneously or after procedures known to induce or stimulate retrovirus production in animal culture systems, including treatment with DMSO, 5'-iododeoxyuridine, corticosteroid, and arginine deprivation. An intracellular cytoplasmic DNA polymerase activity was observed that banded on a sucrose equilibrium density gradient at I .I6 g/ml, the characteristic density of type-C retroviruses. However, as described in the section on cellular enzymes, this polymerase activity had propertieq of D N A 7, not reverse transcriptase.""' No other evidence of intracytoplasiriic virus-like particles was observed by electronmicroscopy or by 3H-adenosine-labeling of cellular RNA. Finally, in tests for possible relatedness to specific retrovirus molecules, including p30-related protein and nucleic acids, no positive results were observed in systems designed to detect homology to probes from woolly monkey (simian) sarcoma virus, _baboon endogenous virus, murine leukemia virus, and feline leukemia virus. * DISCUSSION This article documents the derivation of the myeloid cell line HL-60 from the leukemic blood cell population of a 36-yr-old female with acute promyelocytic HL- leu CUI anc 1 my rep con the the nor mot fror lulo stirr mYe sou I size HLas 1 char of t h TI assol asyn 3).sR have leukc sive cistei parti disse lurn, lcuke intra; essen tliron are rc lcuko* In comrr cliron evider E-gro certifi arm. 15. Pc long a ET AL. -es - - or by Also, )r by lively ys or e for been acid have xpes le of es in d or Lular ,usly imal Me.rase 1.16 d in A7 9 -like < of firus vere ikey and the ytic HL-60 IN ACUTE PAOMYELOCMIC LEUKEMIA 729 leukemia (APL) by demonstrating virtual cytologic identity of the fresh and cultured leukocytes and by defining distinctive chromosomal markers in the fresh and cultured cells. In contrast to cultures of peripheral blood leukocytes from other cases of myelogenous leukemia that we have the HL-60 cells were able to sustain replication in suspension culture in the absence of a continuous added source of conditioned niediuni (i.e., after culture initiation). This does not appear to be due to the secretion of an endogenous growth stimulator, since conditioned medium from the cultured IJL-60 cells did not stimulate the growth of leukemic leukocytes or normal boiic iiiarrow in liquid suspension or semisolid medium. Similar autono- mous growth was noted with all clones of HL-60 cells derived from colonies picked from mythylcellulosc. Further, the HL-60 cells formed colonies in either methylcel- lulose or agar in thc absence of any added source of supplemental growth stimulator. Nevertheless, like fresh myeloid progenitors from some cases of acute myelogenous the cultured HL-60 cells can respond to an exogenous source of colony-stiiiiulating activity (CSA), since the plating efficiency and colony size are greatly enhanced by CSA (Ruscetti et al., in preparation). Finally, the HL-60 cells formed localized subcutaneous tumors in athymic nude mice as early as the 15th iii vitro p;issage (when first tested; Table 5). These properties are characteristic of progressed malignant leukocytes" and establish the oncogenicity of the HL-60 cells iii vivo. The culturcd HL-60 cells have certain cytologic properties that have been associated with leukemic promyelocytes. These include nuclear/cytoplasmic asynchrony, iiuclear bleb formation, and heavy azurophilic granulation (Figs. 2 and 3).5u*JH9owever, both the fresh and cultured HL-60 cells lack other features that have been described for promyelocytes from some cases of acute promyelocytic leukemia, iricludiiig inorphologically abnormal granules with Auer rod and exlen- sive intracytoplasmic libril formation and deformation and dilatation of the cisternae of h e endoplasmic r e t i c u l ~ m T. ~he~ ~la~tte~r cytologic features, have particularly been associated with cases of APL in which hemorrhage due to disseminated intravascular coagulation (DIC) has been prominent.5Bs5T9his, in turn, has been related to the presence of high amounts of tissue thromboplastin in leukemic promyelocytcs.w Although the present patient ultimately died from an intraabdoniinal hemorrhage associated with DIC, her coagulation parameters were essentially normal until terminally. Our preliminary studies indicate that some thromboplastin activity is present in the HL-60 leukocytes; however, further studies are required LOquantitatively relate this thromboplastin activity to that present in leukocytes from other patients with APL and DIC. I n addition to specilic cytologic alterations, APL has been associated with a common karyotypic abnormality involving a reciprocal translocation between chromosomes 15 and 1 7.6'In both the fresh bone marrow and cultured HL-60 cells, evidence was obtained in most metaphases for the presence of a submetacentric E-group-like chromosome. Although the identity of this chromosome could not be certified, it could be a chromosome 17 with extra chromosomal material on its short arm. This chroniatin did not appear to arise by a translocation from chromosome 15. Possibly, i t could have originated from one of the heavy bands of the short or long arms of the inissing chromosome 5.The loss of chromosome 8 in the fresh and 730 GALLAGHER ET AL. cultured HL-60 cells is also interesting, since abnormalitics involving this chromo- some, particularly trisomy, have frequently been associated with human acute Ie~kemia.6**~~ DNA herpesviruses have been associated with the transfornialion of human leukocytes and immortalization of lymphoblast cells in vitro."*6JR N A retroviral information has also been associated with some fresh and cultured huiiian leukemic cells, especially those of myeloid cell and recent experiments in our laboratory suggest that some oncogenic primate retroviruses may have the potential of transforming human leukocyte^.^^ However, no evidence of information related to known herpesviruses or retroviruses has been found in fresh or cultured HL-60 cells. A remarkable feature of the cultured HL-60 cells is their capacity to terminally differentiate despite their aggressive malignant growth potential in vitro and in vivo. This suggests a dissociation of genetic control elements for replication and commitment to differentiation. Such a dissociation has also been suggested for murine (Friend) erythroleukemia cells.` In mouse myelogenous lcukcinia cultured cells, the capacity for myeloid differentiation has been related to alterations in numbers of specific chromosomes." So far, the differentiated phenotype of the HL-60 cells has remained quite stable (see Table 1) despite progrcssive adaptation of the cells to continued culture and despite clonal selection either spontaneously in suspension culture or by deliberate cloning in methylcellulose. This suggests that the observed cytogenetic abnormalities are not directly involved with the differen- tiative capacity of the cells. Certainly, however, phenotypic variations could be present in different clones that have not been detected by the limited niethods so far utilized to characterize the state of differentiation of the HI,-60 cells. In this regard, the differentiative capacity of HL-60 cells appears to diKcr from normal neutrophils, since completely differentiated segmented neutrophils arc rarely seen even after chemical induction" and since at least one enzyme cliaracteristic of normal granulocytes, alkaline phosphatase, was not histochemically detectable in HL-60 cells. Thus, this human cell line seems of unusual interest for studying intrinsic and extrinsic elements that may affect or perturb the processes of myeloid growth and differentiation. ACKNOWLEDGMENT We thank the following persons for their contributions to the studies prcscnted in this manuscript: Dr. L. Glick. Associated Biomedics Systems. Buralo, N.Y.for rosette tcsls and EBNA ;Issays; Drs. M. Reitz, R. Smith, and F. 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