Proc. Nadl. Acad. Sci. USAVol. 91, pp. 1198-1205, February 1994

Review

Cytokine therapeutics: Lessons from interferon a(tumor suppreuor/diferentlation/antivral/anglgene s/cytoskeleton)

Jordan U. GuttermanDepartment of Clinical Immunology and Biological Therapy, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030

ABSTRACT Cytokines are solubleproteins that allow for communication be-tween cells and the external environment.Interferon (IFN) a, the first cytokine to beproduced by recombinant DNA technol-ogy, has emerged as an important regula-tor ofgrowth and differentiation, affectingcellular communication and signal trans-duction pathways as well as immunologi-cal control. This review focuses on thebiological and clinical activities of the cy-tokine. Originally discovered as an antivi-ral substance, the efficacy of IFN-a inmalignant, viral, immunological, angio-genic, inflammatory, and fibrotic diseasessuggests a spectrum of interrelated patho-physiologies. The principles learned fromin vivo studies will be discussed, particu-lady hairy cell leukemia, chronic myelog-enous leukemia, certain angiogenic dis-eases, and hepatitis. After the surprisingdiscovery of activity in a rare B-cell neo-plasm, IFN-a emerged as a prototypictumor suppressor protein that repressesthe dinhl tumorigenic phenotype in somemalgancies capable of differentiation.Regulatory agencies throughout the worldhave approved IFN-a for treatment of 13malignant and viral disorders. The prin-ciples established with this cytokine serveas a paradi for future development ofnatural proteins for human disease.

Molecular biology has revolutionized thestudy and treatment of human disease.Among the many scientific advanceshave been the elucidation of normal cel-lular physiology and pathophysiologyand the ability to produce purified pro-teins that regulate cellular function. Theextracellular signaling molecules knownas cytokines activate a cascade of intra-cellular pathways that regulate growthand differentiation and that produce an-tiviral and immunological responses. Cy-tokines include interferons (IFNs), inter-leukins (ILs), peptide growth factors,colony-stimulating factors, and growthinhibitory and differentiation factors (1).The discovery of IFN in the 1950s led

to the expectation that viral diseasescould be treated (2). Interest beyond thefield ofvirology occurred during the early1960s when workers began to define thesubstance's growth inhibitory and im-mune activation properties (3-5). Be-

cause this species-specific molecule wasdifficult to produce, the field languisheduntil production methods were devised.The most significant of these techniqueswas developed in Finland (4, 6). Duringthe 1960s and early 1970s, reports ofantiviral and antitumor activity in labo-ratory animals and humans stirred up thefield (3, 4). Enthusiasm intensified whentwo groups successfully cloned IFN-aand brought the purified protein to theclinic in 1981, one of the first naturalproteins to become available (7, 8).Three major classes of IFN can be

described: IFN-a, IFN-/3, and IFN-y (9).IFN-a is part of a multigene family, andIFN-P and IFN-y are linked to singlegenes. Because IFN-a and IFN-# sharecomponents of the same receptor, theyare referred to as type I IFNs. IFN-yusesa separate receptor system and is re-ferred to as a type II IFN. AdditionalIFNs have been discovered, but they arenot as well characterized (9).IFNs, which do not normally circulate

and are thought to be formed constitu-tively by most cells, function physiolog-ically by autocrine or paracrine mecha-nisms (10). During stress, the body cancreate large quantities for short periodsof time, and this production is often as-sociated with some toxicity. Several re-cent papers have detailed both precinicaland clinical aspects of the developmentof IFN-a (2-4, 11). This review focuseson important clinical models and princi-ples that serve as guidelines for futureadvances in cytokine therapeutics, espe-cially with respect to regulators ofgrowthand differentiation. Based on these inves-tigations, IFNs have applications wellbeyond virology, including cell biologyand immunology.

Human Cancer: IFN-a CausesRegression of Established Tumors

We began the study of partially purifiedIFN-a from Finland in 1978. The hypoth-esis we hoped to prove was that a natu-rally occurring protein could induce re-gression of established tumors. At thistime, three approaches to cancer therapyexisted, namely, surgery, radiation, andchemotherapy. Our interest in IFN wasstimulated by the clinical investigationsat the Karolinska Institute that suggested

that the impure IFN could delay recur-rent growth of tumor in osteogenic sar-coma patients after surgery (4). In ourfirst study, we learned that pharmaco-logic doses of partially purified IFN-acould bring about regression of tumors ina significant number ofpatients with met-astatic breast cancer, low-grade lym-phoma, and multiple myeloma (12). Re-markably, a natural protein had causedtumors to shrink. Inspired by these find-ings, we began investigations with puri-fied IFN-a2a in 1981. This was the firststudy of a recombinant cytokine, andIFN-a2b became available later thatyear. For the most part, the biologicalactivity seen with the partially pure formwas reproduced with the DNA-derivedversions (13). At the same time, nonre-combinant preparations of purifiedIFN-a containing multiple subtypes wereproduced and developed for the treat-ment of human diseases (4, 6). Pyroge-nicity of the purified IFN-a suggestedthat the febrile response during viral in-fections was due to the endogenous re-lease of IFN and perhaps other cyto-kines. The purified protein was adminis-tered subcutaneously or intramuscularly,and the resulting pharmacokinetic profilewas identical to that reported earlier withthe impure material, which necessitateddaily or every-other-day administration(13, 14).The term cancer describes a collection

of >200 types of malignant neoplasms(15). The abnormal population of cellsconstituting a malignancy demonstratestemporal unrestricted growth preferenceover its normal counterparts, inhibitionof differentiation, and the tendency toinvade tissue and metastasize (15). Ad-vances in molecular biology have shownthat the development of certain tumors isrelated to mutations of oncogenes thatregulate growth and differentiationand/or to mutations or loss of tumorsuppressor genes (16). Classically activechemotherapeutic agents are defined by

Abbreviations: CML, chronic myelogenousleukemia; HCL, hairy cell leukemia; HIV,human immunodeficiency virus; IL, interleu-kin; IFN, interferon; MHC, major histocom-patibility complex; TH, T helper; EBV, Ep-stein-Barr virus; HBV, hepatitis B virus;HCV, hepatitis C virus.

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their ability to induce regression oftumorin a percentage of patients with a partic-ular tumor type. Generally, the rule is aminimum of 15-20% of patients mustrespond. In the early 1980s, the partiallypurified and the recombinant IFNsshowed various degrees of efficacy assingle agents to induce tumor regressionin malignant blood and solid tumors (4,12, 13), but this ability was not sufficientto gain federal government approval formarketing in the United States. We thenbegan to look for novel clinical targetsthat could serve as a guideline for furtherdevelopment of this growth regulatorymolecule.Hematologic Malignancies: Lessons

from Hairy Cell Leukemia (HCL). Basedon the ability of IFN-a to induce remis-sions in some patients with well-differen-tiated B-cell tumors (4, 12), we decided toinvestigate the protein in a rare B-cellneoplasm known as HCL. In 1982, noeffective treatment existed other thansplenectomy. With IFN-a, the sup-pressed levels of peripheral blood cellsand platelets rapidly increased (17). Theimmune status improved and hairy cellsin the bone marrow and blood declined.For these patients, the incidence of op-portunistic infections disappeared andthe requirement for platelet and erythro-cyte transfusions abated. Low doses ofthe therapy were effective, and endoge-nous IFN-a production was restored tonormalcy after remission was achieved(18). After the presentation of this work,the activity was confirmed rapidly withthe recombinant molecule (19), dissipat-ing much of the skepticism regarding acytokine's potential in human therapy.These studies led to approval in June1986 by the U.S. Food and Drug Admin-istration, and 31 other regulatory bodiesthroughout the world soon followed (14).Recently, chemotherapeutic agents havesupplanted part of the role of IFN-a intreating HCL, but the principles of dis-covery leading to regulatory approvalhave served as an important paradigm forcytokine development.The precise mechanism of HCL's

unique sensitivity to IFN-a is unknown,but differentiation, cell-cycle arrest,and/or apoptosis may play roles in thecompromised survival of the malignantcells (20). Normal B-cell growth and dif-ferentiation follow an established path-way, and malignancy may occur at anystage ofdifferentiation and freeze the cellat that point, immortalizing it (21). Thehairy cell is a well-differentiated pre-plasmatic B cell that expresses the CD25(tac) antigen. The presence of the CD25antigen and its growth factor, IL-2, ap-pears to be important since variants ofHCL that lack the CD25 antigen areresistant to IFN (20). With IFN-a ther-apy, the decreased survival of the malig-nant hairy cell leads rapidly to the resto-

ration of normal hematopoiesis (17, 19,20). The most compelling evidence seemsto suggest that IFN-a provides an anti-growth signal to allow completion of dif-ferentiation and/or growth arrest. In ad-dition, by blocking various B-cell growthfactors, IFN may allow normal matura-tion to continue (20).

Additional clues as to how IFN-a maywork in HCL are provided by the highintracellular levels of free calcium andthe phosphorylation of CD20, a cell sur-face molecule (22). The phosphorylationof CD20 relies on calcium/calmodulin-dependent protein kinase II and proteinkinase C. IFN-a downregulates the phos-phorylation of CD20 and decreases thehigh levels of intracellular calcium inleukemia cells (22). Protein kinase C,which has a negative effect on calcium/calmodulin-dependent protein kinase II,also may be activated by IFN-a (23).Interestingly, the CD20 molecule hasstructural similarity to Epstein-Barr vi-rus (EBV) latent membrane protein 1, atransforming oncogenic virus protein(24). EBV latent membrane protein 1turns on bcl-2, a gene that prevents apo-ptosis. Since bcl-2 is expressed in HCL,IFN may be working by indirectly down-regulating the gene and allowing apopto-sis to occur; however, other nonnecroticmechanisms of tumor cell death may beoperating (22). Another oncoproteinhighly expressed with HCL is c-Src (25),a protein-tyrosine kinase that may bedampened by IFN-a (5). A link may existbetween abnormal tyrosine kinase, highintracellular calcium levels, CD20 phos-phorylation, and blocked differentiation(22).Other B-cell neoplasms also show var-

ious degrees of sensitivity to IFN-a (3, 4,11, 12). In patients with multiple my-eloma and low-grade lymphoma, the cy-tokine often has demonstrated a positiveclinical impact on survival when com-bined with chemotherapy (26-29). It maybe postulated that even more significantsurvival benefit may result if IFN-a wereto be administered chronically and con-tinuously rather than in short pulses.When given early in the disease, remis-sions can occur in chronic lymphocyticleukemia, indolent myeloma, and cryo-globulinemia, and suppressed immuno-globulin production can be restored to-ward normal levels (14, 30, 31).

Lessons from Chronic MyelogenousLeukemia (CML). Clinical research withIFN-a in patients with CML has pro-vided important clues to the potentialfuture application of cytokines in cancerand other diseases. CML is a multilin-eage hematologic malignancy, character-ized by the presence of the genetic aber-ration known as the Philadelphia chro-mosome (32, 33). The chromosomeresults from a reciprocal translocationbetween chromosomes 9 and 22, which

forms the chimeric bcr-abl oncogene.The protein product shows an elevatedtyrosine kinase activity when comparedto normal c-Abl protein. This progressiveneoplasia originates from a clonal out-growth ofpluripotent hematopoietic stemcells. During the chronic or benign stage,the peripheral granulocyte level increasesand the spleen enlarges (33). The cloneretains the capacity to differentiate into afunctional granulocyte, which distin-guishes it from the immature cells seen inacute leukemia. CML usually progressesto undifferentiated leukemia, character-ized by circulating undifferentiated my-eloid or lymphoid blast cells.Although a fraction of CML patients

receiving bone marrow transplantationcan be cured, chemotherapy alone hasnot had an impact on the prognosis (33).Based in part on in vitro effects of IFN-aon myeloid progenitor cells (34), our in-vestigation of the role of the cytokineduring the chronic phase of CML dem-onstrated that it could cause hematologicremission. Approximately 75% of pa-tients with benign phase achieve a com-plete normalization of blood counts. Fol-low-up examinations revealed that IFNhad the astonishing capacity to suppressselectively cells bearing the Philadelphiachromosome, resulting in partial or com-plete restoration of the normal clone.This work was confirmed rapidly withrecombinant IFN-a (35), and cytogeneticanalysis revealed that 20-25% of patientsexperienced sustained elimination ofcells bearing the Philadelphia chromo-some. These data have been confirmedand expanded (36). Whether the malig-nant cells are completely eliminated orsuppressed awaits further analysis (37).Many of these patients have durable re-missions lasting up to 8 years (36). Sur-vival of such patients may be improvedwith prevention of transformation to theblast phase of the disease. Although thefull impact ofIFN-a in patients exhibitingcomplete resolution of the Philadelphiachromosome is still under debate (38), amajor biological effect with potentiallyimportant clinical significance has beenachieved.Minimal effects of IFN-a are seen in

the accelerated or acute phase of CML.As the disease advances, additional cy-togenetic abnormalities occur and auto-crine growth factors are produced (39-41). These factors can account for intrin-sic resistance or escape from growthinhibition. One of these factors, IL-1f3,can be antagonized in vitro with the sol-uble receptor or the IL-1 receptor antag-onist, which is shown to be additive withIFN-a in suppressing the growth ofCMLprecursor cells (41).

Parallel with the clinical investigationhas been an explosion of knowledge ofthe molecular and biological events inCML (42). Important murine models of

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CML have been developed, and the fail-ure to transplant the cells from the benignphase ofthe disease suggests that tumorsoriginate from committed myeloid pro-genitor cells that undergo clonal expan-sion but are not fully transformed (43).The efficient transplantation in the latestages suggests full transformation.Thus, the early stage-represented asdiscordant maturation rather than excessproliferation (33)-is biologically distinctfrom the later stages.One way that IFN may compromise

the survival of the malignant CML cell isby downregulating the expression of bcr-abl (44). Before therapy, the expressionof the Bcr-Abl protein may prevent apo-ptosis (45), thus allowing for accumula-tion and excess survival of the CML cell.Data show that the ber-abl gene productlocalizes to filamentous actin (46), whichlikely results in abnormal communicationthrough the cytoskeletal network, con-tributing to transformation of cells (47).The signal transduction pathways of bcr-abl are being worked out; the oncopro-tein attaches to Grb-2 protein and prob-ably involves ras pathways (48). Theabnormal accumulation of calcium mayplay a role in CML since irregular tyro-sine kinase activity and high levels ofcalcyclin-a calcium-binding S100 pro-tein involved in cell-cycle progression,differentiation, and possibly cytoskeletalinteractions-are observed (49, 50). Inother systems, IFN suppresses ras trans-formation in vitro, possibly by inducinglysyl oxidase, a protein involved withcollagen and elastin formation (51, 52).Associated with the Philadelphia chro-mosome is a defect in the CML cell'sadhesiveness to stroma, which can benormalized with IFN-a (53). IFN-a mayrestore adhesion by upregulating lym-phocyte function-associated antigen 3(CD58), a glycosyl-phosphatidylinositol-anchored protein (54), or may affect thefunction of other adhesion molecules,such as f31 integrin (55), which is con-nected to actin.

Certain tumors of the myeloid systemthat are partially differentiated, such aspolycythemia vera and disorders involv-ing high platelet counts (14, 56), also aresensitive to IFN-a. In contrast, tumorsthat are resistant include the undifferen-tiated acute myeloid leukemias and my-elodysplastic syndrome, a disorder ofmyeloid maturation. In one variant ofmyelodysplastic syndrome, a transcrip-tional factor induced by IFN is involved(57). Important molecular differences be-tween acute and chronic leukemias havebeen discussed (58).

Solid Malignancies and Other Angio-genic Diseases. As in certain hematologicmalignancies, solid tumors progress in

stages, with increasing mutationalchanges, eventually leading to autono-mous growth (15, 16, 21). Progression

relies on a number of biological pro-cesses. At first glance, the results ofIFN-a therapy in solid tumors mightseem discouraging; with few exceptions,minimal activity is observed in far ad-vanced epithelial and mesenchymal can-cers (4, 11, 14). However, certain solidtumors in the metastatic stage, such asslowly proliferating variants of renal cellcarcinoma and malignant melanoma, un-dergo regression in a small fraction ofpatients, perhaps 10-15% (4). Neuroen-docrine tumors ofthe gut have respondedto IFN-a also. The most illustrative datacome from carcinoid tumor, where themajority ofpatients have improvement ofsymptoms and a smaller fraction undergotumor regression (59). Ultraviolet-irradiation-associated cutaneous tumorsare emerging as important models for theunderstanding of biological modulationof malignancy. Both squamous and basalcell carcinomas of the skin show sensi-tivity to IFN-a, as a single agent or incombination with retinoids (60, 61). My-cosis fungoides, a malignancy of the Thelper (TH) cell that may be stimulatedby IL-7 as an autocrine factor (62), is alsosensitive to IFN-a alone or with othermodalities, including retinoids (63, 64).IFN-a-induced regulatory factors maycontrol the IL-7 receptor expression (65).Retinoids may operate by affecting an-giogenesis (66), the cytoskeleton, or pos-sibly by modulating cytokines such astransforming growth factor 3 (TGF-,B),which is important in the control of ke-ratinocyte growth and differentiation(67).The majority of studies with metastatic

cancer have been carried out after con-ventional therapy has failed and the tu-mor has undergone multiple mutationalchanges. Based on the principles devel-oped in CML, it is highly likely that IFNapplied early in the stages of tumor evo-lution could have a very important clin-ical effect, whereas its activity in ad-vanced stages would be minimal. Theseprinciples are being examined in twosolid tumor models, and the results areencouraging. One model is osteogenicsarcoma, a mesenchymal-derived tumorassociated with a high frequency of Rband p53 mutations. Although long-termbenefits are still unclear, the studies con-ducted with IFN-a after surgery providea blueprint of possible value for early useof a cytokine in a clinically relevant stage(68). Studies with nude mice suggest animportant effect on tumor cell differenti-ation (69). Another model that may serveas a guideline for future studies is primaryhigh-risk malignant melanoma, a neuro-endocrine malignancy. The biological ba-sis for tumor growth and progression inmelanoma has recently been presented(15, 70, 71). Progression is correlatedwith resistance to cytokines (70). IFN-aappears to delay recurrence and prolong

survival after surgery (72). This workconfirms the notion from HCL and CMLstudies that chronic maintenance ther-apy, perhaps lifetime, may be required toprevent recurrent disease. It is likely thatthese principles are relevant for epithelialcarcinomas. Many opportunities exist totest these ideas.

Angiogenesis, a crucial step in thetransition from hyperplasia to neoplasia(73), is an exciting target for therapeuticintervention in early malignancies likeprimary melanoma (74), at a stage whenIFN might have the most dramatic ef-fect. Metastasis of solid tumors dependson angiogenesis and other biologicalprocesses of invasion (75, 76). The pro-posal that inhibiting neovascularizationcould be useful as an anticancer therapy(73) is supported by the fact that tumorsuse angiogenic molecules to induce newblood vessel formation from host ves-sels. The number of microvessels in theareas of invasive breast carcinoma andother tumors predicts the likelihood ofmetastatic progression (73). Several dis-eases beyond cancer are characterizedby abnormal growth of new capillaries(73, 77). At least seven angiogenic mol-ecules have now been identified, as wellas numerous angiogenesis inhibitors(73, 78). IFN-a is the first of the knowninhibitors (3, 78) to be evaluated clini-cally. In addition to melanoma and thetumors mentioned above, IFN-a hasshown its potential in other angiogenicdiseases. Kaposi sarcoma is an angio-proliferative disease that commonly de-velops in individuals infected with thehuman immunodeficiency virus (HIV)(79). The early studies of recombinantIFN-a demonstrated that 40% of pa-tients experienced significant regressionof lesions (4). This work led to theapproval ofIFN-a by the U.S. Food andDrug Administration and 21 other coun-tries for the treatment of AIDS-relatedKaposi sarcoma (14). More recently, theinduction of dramatic regressions byIFN-a in children with life-threateningpulmonary hemangioma has revealedfurther the benefits of an antiangiogenicagent (73).Other angiogenic diseases represent

important targets for IFN-a; however,multiple steps in angiogenesis may haveto be approached simultaneously to ef-fectively block all aspects ofblood vesselformation (66, 78). The development ofmolecules that can synergize with IFN-ain preclinical models is encouraging (78).Diagnostic tests for angiogenic activitywill be important to guide therapy, par-ticularly for early cancers (80).

Discussion: The Emergence of TumorSuppressor Proteins. Cancer is a disorderof cellular development, characterizedby impaired differentiation and dysregu-lated growth (15, 16, 81). It is usually achronic disease (82) that progresses

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through various genetic stages leading toautonomous, often undifferentiatedgrowth and/or a metastatic phenotype(16, 21, 83, 84). While cancer is a diseaseof the tissue (66) with disrupted cell-to-cell communication and an irregular ex-tracellular matrix (47, 85-87), systemictherapy-primarily chemotherapy-generally has been directed at tumor cellreplication using cytotoxic drugs thatdamage DNA or other components ofthemalignant cell. As the biological and ge-netic lesions of cancer come into sharperfocus (16, 88-92), the list of potentialtargets for tumor control expands to in-clude the extracellular matrix (81), tumorstroma, cell motility mechanisms, celladhesion molecules, blood vessels, cyto-skeletal network (47), signal transductionpathways (93), as well as translational(94) and transcriptional (16) events.IFN-a has emerged as an exciting pro-

totypic tumor suppressor protein withimportant properties that regulate normaland malignant cell growth and differenti-ation (95). The antigrowth effects involvemultiple pathways and include inductionof Go/G1 arrest, suppression of Rb phos-phorylation, downregulation of G1 cy-clins and cyclin A, and inhibition of E2FDNA binding and c-myc expression (%).IFN antagonizes mitogenic activity oftissue and hematopoietic growth factors(5, 97) and downregulates function ofcytoplasmic tyrosine and serine-threo-nine oncoproteins involved in signaltransduction (5). The interesting obser-vations on inhibition of cell motility (5,208), increase in cell surface fibronectin,as well as the reordering of disruptedcytoskeleton, especially actin filaments(5), are becoming increasingly relevant.Cell motility is critical in the biology ofmetastasis and can be stimulated by pro-teins such as acidic fibroblast growthfactor and IL-6 (87, 208). The effect ofIFN on some cell adhesion molecules(54, 55) and their importance in cellularbehavior (1, 98, 99) stimulate the idea thatcadherins and the junctional plaque cat-enins (100-102) might be regulated bythis cytokine as well. This seems likelybased on the in vitro effects of IFN-a onsrc-transformed fibroblasts (5) wherecadherin function is abnormal (101). In-terestingly, IFN regulatory factor bind-ing sites have been identified within thepromoter regions of the E-cadherin gene(103).

Signal transduction pathways are tar-gets for IFN's antitumor action. Mitosisand other key metabolic events are reg-ulated by Ca2+ (104, 105). The emergingevidence that IFN may regulate Ca2+metabolism in HCL may be pertinent toCML, melanoma (106), and other malig-nancies (50). Other likely targets for reg-ulation by IFN include cytoskeleton-associated proteins whose expressioncan suppress tumorigenicity, such as

a-actinin, tropomyosin, vinculin, and vi-mentin (107, 209). It will be of interest todetermine whether phosphorylatedCD20, like phosphorylated EBV latentmembrane protein, disrupts vimentin in-termediate filaments. In fact, IFN in-creases expression of vimentin in differ-entiated tumor cells and fibroblasts (209).Also, IFN prevents the loss of vinculinfrom adhesion plaques in growth factor-treated cells (208). Effects on other mi-crofilament-associated proteins such asmyristoylated alanine-rich C kinase sub-strate (MARCKS) and caldesmon, inter-mediate filament proteins, and microtu-bles need to be studied (47, 108). IFNinduces expression of an actin monomerbinding protein, thymosin 14 (109). Fur-thermore, in HCL cells, where the cy-toskeleton is disrupted (20), IFN-a in-duces the reorganization of spectrin(110), a src homology 3 domain-contain-ing structural protein (108). In CML andother tumors, further investigation isneeded on IFN's regulation of src homol-ogy 2 and src homology 3 domain signal-ing events (48) as well as ras pathways,either involving cell division or cell mor-phology and the actin cytoskeleton (111).Of interest is the recent evidence thatIFN-a blocks Raf-1 phosphorylation invitro (112). Finally, since ras ultimatelyleads to activation of myc (111), the in-hibitory effects of IFN-a certainly in-volve transcriptional events (96) andgene expression (47).The most important clinical application

to date in cancer involves the slowlyproliferating well-differentiated tumors.The maximal response to IFN-a therapy,shown by partial or complete suppres-sion of the malignant clone, may require12 months or longer. To maintain thetumor cells in a quiescent or dormantstate, as for example, by blocking angio-genesis (73), chronic long-term adminis-tration seems to be required. The precisemechanisms of compromising tumor cellsurvival (for example, in HCL and CML)are not clear, but they seem to involvenonnecrotic pathways. Effects on tumorcell differentiation (81), induction of apo-ptosis (113, 114), or possibly senescence(115) may be operating in different sys-tems. The latter is an interesting possi-bility based on the recent link between atumor suppressor protein and senescence(116). Based on the cell cycle data dis-cussed above (96), IFN-a possibly in-duces the expression of this protein (116).Immunological mechanisms may be im-portant (2, 117, 118), particularly withsmall tumor volume. Of particular inter-est is the ability of IFN-a to enhancemajor histocompatibility complex(MHC) class I antigen expression andinduce gp%, a heat shock protein asso-ciated with antigen presentation (119).The surprise is that a single suppressantmolecule can have an important impact in

several tumors early in evolution. Thedata suggest, however, that two or moretumor suppressor proteins or regulators,such as IFN-a in combination with retin-oids, will be required to overcome resis-tance often associated with mutant sup-pressor genes or overexpression of on-cogenes (67, 89, 96) to allow theelimination of the malignant clone.Although the molecular events of tu-

mor progression may differ in the chronichematologic malignancies, compared todeveloping tumors of the mesenchymal,epithelial, and neuroendocrine tissues(120), we speculate that intervention atan early stage of cancer might have aprofound clinical impact whereas little orno effect is seen in the late stages, whichhave multiple genetic aberrations. As tu-mor cells accumulate and fail to die, evenwith low growth fractions as demon-strated in CML, HCL, and early mela-noma, they are increasingly susceptibleto additional mutational changes (83, 84)leading to tumor progression (21). Thus,a decline in tumor-cell survival (121, 122)with restoration of normal cell growthcould prevent progression.

Differentiation of normal tissues is aplastic process that requires continuouscontrol (123). In cancer, tumor suppres-sion is linked to growth control and dif-ferentiation (81). The studies described inthis essay reveal that, for certain tumorsstill capable of differentiating, the malig-nant process potentially is reversible al-lowing for normal cellular differentiation.This idea would verify the in vitro con-cept of reverse transformation (5, 47, 52,124), where genetic reprogramming, re-quiring an intact cytoskeleton (47), oc-curs.

Viral Diseases

The body protects against invading vi-ruses in a multitude of ways, and recentgene knockout experiments in mice illus-trate the critical role that both type I (M.Aguet, personal communication) and typeII IFNs play in defense against viruses(125). IFN-a induces an array of potentproteins regulating viral and cellulargrowth (126). In addition, IFN-a activateskey components of the cellular immunesystem important in viral recognition (11,14). Despite this natural defense, someviruses are adept at subverting the IFNresponse, including adenoviruses, certainherpes viruses (particularly the EBV),hepatitis B virus (HBV), and HIV (126,127). During the 1970s, several viral in-fections showed promise in pilot studieswith the partially purified IFN-a. Theavailability of the purified IFNs resultedin the expansion of these studies, andtoday, IFN-a is the treatment of choicefor patients with chronic HBV and hepa-titis C virus (HCV) infections (11, 14).

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HBV. After treatment with IFN-a for 6months, 40% of patients experience aclinically useful response (11, 128). Ele-vated liver function is normalized innearly all patients who show a sustainedloss of HBV DNA and the HBV e anti-gen. Recurrence is rare once a responseto IFN is observed. The HBV surfaceantigen can disappear mopths or yearsafter the cessation of therapy in 15% ofpatients, leading to the termination of thecarrier state (128). This elimination ap-pears to be mediated by the cellular im-mune system. In July 1992, the U.S.Food and Drug Administration approvedIFN-a2b for the treatment of chronichepatitis B. Nineteen countries have ap-proved the use of the cytokine for thisindication (14).

Certain viral mutations and host re-sponses are thought to explain the casesunresponsive to IFN-a (129). The hostresponses leading to chronic hepatitisinclude impaired IFN production and aninhibited cellular response to IFN due tothe hepatitis B terminal protein. The sig-nificant fraction of patients showing re-sistance illustrates the need for furtherstudies with a combination of agents.A link has been demonstrated between

chronic HBV infections and hepatocellu-lar carcinoma (130). Since the carcinomaappears years after the onset of chronicliver disease, inflammation with genera-tion of oxygen free radicals (84), hepato-cyte regeneration, cirrhosis, and thetransactivation of hepatitis X protein areall thought to contribute to the develop-ment of the malignancy. Some of theseevents may be controlled by IFN.HCV. Previously known as non-A,

non-B hepatitis, hepatitis C is sensitive toIFN-a in -50%o of patients (131). Ten to25% undergo sustained responses with arestoration of normal liver functions.IFN-a has been approved for the treat-ment of chronic hepatitis C in 10 coun-tries (14). Remissions can be reinduced inpatients who relapse upon reintroductionof IFN-a therapy.

Certain strains of HCV are relativelyresistant to IFN, and data suggest thatpatients may be infected with multipleviral subtypes (132, 133). A need existsfor agents that can potentiate the activityof IFN-a. One such agent is N-acetylcysteine, an enhancer of glutathione pro-duction that may have relevance for HIVinfection as well (134). Also effective forchronic hepatitis C is ribavirin (131),which has activity in certain persistentviral infections of animals (135).Whereas increasing evidence shows

that the HBV-specific cytotoxic T cellscan mediate liver cell necrosis and con-tribute to the pathogenesis of hepatitisB-associated liver cell injury, only re-cently have HCV-specific cytotoxic Tcells been found within the liver to pro-vide a means to evaluate their possible

pathogenicity and role in elimination ofthe virus (136). Epidemiological studiessuggest that infection with HCV, which isan RNA flavi- or pesti-like virus, caneventually progress to the developmentof hepatocarcinoma.HIV. IFNs play a key defense against

HIV (137, 138); as a therapy, IFN-a maybecome important, particularly when ad-ministered early in the course of thedisease (139). Since lymph nodes providethe retrovirus with a reservoir (140),IFN's ability to traffic lymphocytes tolymph nodes, in part, by inducing theL-selectin receptor (210) and to interferewith cell-to-cell HIV transmission maybe future avenues of intervention (141).The cytokine's antiviral influence may bemediated through a variety of mecha-nisms, including in vitro inhibition of theRev protein (142). The demonstrationthat IFN can preferentially stimulate invitro the TH cells, subtype 1 (TH-1 cells)is of interest (143). Based on the sugges-tion that TH-2 cells partially may beresponsible for progression in AIDS(144), studies with IFN in early HIVinfection are being pursued. Paradoxi-cally, as the disease progresses, activa-tion ofIFN-a may signal a poor prognosis(138).

Papilloma Virus. A certain subset ofthe >60 types of human papilloma vi-ruses, a family of DNA viruses, are as-sociated with epithelial tumors (130). Forexample, human papilloma virus types 16and 18 are associated with malignant cer-vical intraepithelial neoplasms, and thecombination of IFN-a and retinoids hasbeen found surprisingly active in thiscondition (145). Human papilloma virustypes 6 and 11 are associated with benigncondylomata acuminata and laryngealpapilloma. Regulatory agencies in 10countries have approved the use ofIFN-a for condyloma acuminata, and 5countries market the protein for juvenilelaryngeal papilloma (4, 14).

Discussion. Clearly, IFN-a is an effec-tive treatment for a variety of viral infec-tions. With the threat of new viral dis-eases (146) and resistant strains of 'rec-ognized viruses, the need exists for moreeffective therapy. Very recently, excitingantiviral effects with IFN-a have beenreported in chronic hepatitis D, a partic-ularly severe disease (164). The notionthat IFN is a tumor suppressor protein aswell as an antiviral substance is consis-tent with the fact that EBV, HBV, HCV,certain papilloma viruses, and humanT-cell viruses are linked with human tu-mors. These viruses use various modal-ities for transforming cells, including in-teraction with suppressor genes, bluntingof the host immune response, and gener-ation of oxygen free radicals as potentialmutagens (84, 130). Disruption of cy-toskeleton by HIV and other viruses hasbeen implicated in the development of

tumors (107, 147). Therefore, IFN's di-rect effect on cell and viral metabolism,as well as its enhancement of T-cell re-sponsiveness, makes it an exciting agentfor the therapy of chronic viral infectionsand possibly modifies the carcinogenicrisk.

Fibrosis

Fibrosis is a normal repair process oftissues after injury (148, 149). The criticalinteraction of growth factors, cytokines,and the extracellular matrix in this pro-cess has been reviewed recently (1, 86,149). IFN-a is involved with this processthrough its effect on collagen metabolism(150) and through inhibition of tissuegrowth factors (5, 97). Numerous condi-tions are associated with dysregulatedfibrosis of various tissues. A recent re-port with the use of IFN-a in chronichepatitis patients demonstrated normal-ization of the biochemical markers offibrosis and mRNA for TGF-,l. Regres-sion in hepatic inflammation occurred(151). A report on regression of keloidformation by IFN-a has appeared (152).Also, patients with keloids were found tohave an IFN deficiency (153). The effectof IFN-a on keloids may be quite rele-vant to tumor stroma, which has beenaptly characterized as a wound that failsto heal (154). A related concept linkingcellular injury, proliferative disorders,and neoplasia has been presented (122,155). Although these events do not in-variably lead to cancer, they may beregulated by an agent like IFN-a.Another possible application is arterio-

sclerosis (156, 157), especially restenosisafter percutaneous translumenal angio-plasty, since IFN-a antagonizes growthfactors responsible for smooth muscleproliferation and has a putative role inmodulating lipids that have antiviral ac-tivity (158). In preclinical models, thetherapy has shown success, as has IFN--y(156, 157). Other candidate diseases in-clude progressive systemic sclerosis(scleroderma), pulmonary fibrosis, ocu-lar fibrosis after glaucoma surgery (159),and possibly ankylosing spondylitis.

TH-1 and TH-2 Cells andInflammatory Diseases

Immunological diseases are being de-scribed by the distribution of the TH-cellpopulations. For example, psoriasis hasbeen characterized by the presence ofTH-1 cells (160). Recent evidence sug-gests that IFN-a, which is important inT-cell development, stimulates produc-tion of TH-1 cells, which primarily makeIFN-y and IL-2, at the expense of TH-2cells, which make TL-4 and IL-S (143).IFN-a, which is known to exacerbatepsoriasis (161), may do so by overstimu-lating the TH-1 cells. Studies suggest that

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keratinocytes bear the anchored lympho-cyte function-associated antigen 3 adhe-sion molecule (CD58) (162), which may-after stimulation by IFN-a-enhance theTH-1 cells to release cytokines, causingprogression of the disease.On the other hand, IFN-a therapy has

been shown to improve a variety of dis-eases characterized by excess TH-2cells, including early AIDS (139), basalcell carcinoma (61, 163), and hypere-osinophilia (165). Allergic diseases due toexcess IL-4 and IgE production or intra-cellular parasitic diseases also may re-

spond to IFN-a (165).Recent observations suggest that IFN-a

may also be useful as an nntiinflammatory

agent. The observations include suppres-sion ofthe chemotactic protein, IL-8 (166),induction of the antiinilammatory IL-1 re-ceptor antagonist (167), and potential util-ity in endotoxic shock (168). The lattermaybe explained by the finding that lethal en-dotoxemia is potentiated by macrophagemigration inhibitory factor (169), which isalso a binding protein for an IFN antago-nist, sarcolectin (124, 170). Preliminary re-

sults suggest IFN-a may be effective inCrohn disease (171), a granulomatous dis-order that has been linked to IL-10 by geneknockout studies in mice (172).

Increasingly, cytokines will be used toregulate each other. IFN-a works withIL-12 in driving cells toward a TH-1phenotype rather than TH-2 (173). Thecurrent use of IFN-I3 in the treatment ofmultiple sclerosis is a good example ofone cytokine regulating others. IFN-Pdownregulates tumor necrosis factor andIFN-y, which are neurotoxic, as well ashuman leukocyte antigen class II, and itupregulates the immunosuppressive cy-tokine TGF-3 (174). In the future, theintricate interactions between cytokines,their receptors, and their inhibitors willform the framework of an entire disci-pline of cytokine biology (175).

Toxicity

The IFNs, like most other cytokines, areproduced by the body to act locally.When used as a systemic pharmacologic,certain toxic effects are seen (2, 4, 14,176). Skin, neurologic, endocrine, andimmune toxicities have been reported.IFN-a therapy may result in fatigue, fe-ver, and weight and appetite loss. Incertain patient populations, the toxicityis negligible. Over the past decade, thetoxicities have provided insight into thephysiological and pathophysiologicalroles of IFN (2).While rarely associated with toxicity,

neutralizing antibodies have developed inpatients treated with certain forms ofIFN-a (13, 177, 178). The antibodies mayabrogate biological effects in vivo. Aminoacid composition might explain the dif-ference in immunogenicity between re-

combinant subtypes. Occasionally, thepresence of antibodies spontaneously re-

solves after long-term therapy.Autoimmune Processes. IFN-a can in-

duce autoimmune processes, and it hasbeen associated with the exacerbation ofdiseases associated with MHC class Iantigens, such as psoriasis (161), andwith MHC class II antigens, such as

thyroid abnormalities (176). Most reportshave concentrated on the induction ofsystemic lupus erythematosus, an exam-ple of multisystem autoimmunity, or au-

toimmune thyroid disease, an organ-specific disorder (2, 4). An acid-labileform of IFN has been extracted from theserum of patients with a variety of au-toimmune disorders (2, 179). This form isalso associated with advanced AIDS andsignals a poor prognosis (138, 179).IFN-a preferentially induces MHC

class I antigens, and lupus has generallybeen associated with MHC class II anti-gens. A recent report showed that micedeprived of the ability to make MHCclass I antigens are resistant to lupusinduction (180). Diabetes in mice hasbeen associated with overproduction ofIEN-a and y (181). Recent data linkingimmunopathology with viruses (182)must also take into account the role ofcytokines, such as IEN. Current con-cepts of autoimmunity have been pub-lished (183).

Neurological Effects. The effects ofIFN-a on the central nervous systeminclude behavioral changes and moderatecognitive, affective, and personality dif-ferences (4, 184). Changes in the highermental functions may be due to a directeffect of IFN on the frontal lobes or thedeeper brain structures. These effectsvary according to such factors as doseand age of patient (176).The immune and central nervous sys-

tems interact (185), and during chronicviral infections, cytokines can influencethe hippocampus and other parts of thebrain. Several growth factors can in-crease the magnitude of long-term poten-tiation induction in the hippocampus(186). The synaptic potentiation in rathippocampus is suppressed by IFN (186).This finding is consistent with the mem-ory impairment reported in many studies.Gene knockout experiments in mice sug-gest that the calcium/calmodulin-depen-dent protein kinase II is important ininducing long-term potentiation and

memory (105). Ifthe suppressive effect ofIEN-a on this enzyme or on Ca2+ metab-olism is operational in neurons as it maybe in hairy leukemia cells (22), then onemechanism for memory impairment seen

in vivo may be due to the inhibition of thepathway, producing nitric oxide (187).The inhibition of P450 reductase (188)and its effect on carbon monoxide isanother possibility (189). Recent trans-genic mouse experiments verify the toxiceffects of IFN in the brain (190).

Perspective

Cytokines are being investigated and em-ployed in a myriad of human disorders.Simultaneously, various disciplines areunraveling the molecular basis of dis-ease, which often involves cytokines.This review underscores the complexi-ties of and opportunities for drug devel-opment. Although much attention hasfocused on the use of growth factors forvarious medical conditions, less attentionhas been given to applications of inhibi-tory or differentiation molecules (191) forhuman pathology characterized by ex-cess mitosis or tissue growth (149, 155,192, 193) and defects in differentiation(81). IFN's activity in inflammatory, fi-brotic, angiogenic, viral, and malignantdiseases suggests a spectrum of interre-lated pathophysiology (Table 1). Thework with IFN-a has opened up manycellular targets in a variety of diseases,where even more specific agents cer-tainly will be developed (90, 194-196)(Table 2). This review, for example, sug-gests a new paradigm for the therapy ofhuman malignancy in that the use of a

tumor suppressor protein can be utilizedto exploit the new biological understand-ing of cancer progression. The questionsclinical researchers pose have changed:In the 1970s, we asked whether IFN-acould induce tumor regression; in 1994,we wonder whether IFN-a may be usefulto prevent carcinogenic changes (197).Can IFN prevent the recurrence of pri-mary solid tumors? Will IFN be syner-gistic with other tumor suppressor pro-teins (116) or regulators of growth anddifferentiation? What are the pathways ofresistance?A great deal of room exists for refine-

ments in our understanding of the IFNsystem. Recent work on the complexitiesof the receptor (198), the signal transduc-

Table 1. Highlighted clinical principles with IFN-a

1. Well-differentiated slowly proliferating malignancies are sensitive. IFN-a inducesquiescence in early malignancies.

2. Rare tumors can be important in drug development and can provide insights intomechanisms of action.

3. Number and type of tumor-associated genetic events may correlate with sensitivity to

tumor suppressor proteins (for example, benign vs. blast phase of CML and primary vs.

metastatic malignant melanoma).4. The spectra of diseases in which IFN shows activity are interrelated.

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Table 2. Important targets for IFN-a1. Extracellular matrix and tumor stroma2. Cell adhesion molecules3. Cytoskeletal function4. Signal transduction pathways5. Cell cycle proteins

tion pathways (199), and the IFN-inducible genes that regulate viruses andcellular growth and differentiation (95),lends itself to pharmacological and bio-chemical manipulation. The effects onmitochondrial metabolism (200) and ubi-quitination (201) need to be clarified. Thebiologic roles of the various IFN-a spe-cies need to be studied. Drug deliverysystems should be assessed, particularlyfor angiogenic and proliferative diseasesaffecting single organs. Dose-responseeffects, in a variety of conditions, andcombinations with other suppressor pro-teins, as well as immunological hor-mones, need to be evaluated. The com-bination with classic chemotherapeuticdrugs, which often work through apo-ptosis (202, 203), and hormonal therapy,as in the combination with retinoids,needs to be expanded. Cytokine biologyis emerging as important as endocrinol-ogy has been in the study ofhormones inthe last 50 years. For example, IFN de-ficiency states may be involved in a num-ber of diseases. With the possible con-nection between cytokines and DNA re-pair (204, 205), the decline in IFN levels,resulting from aging or environmentalinsults (206), may be involved in thepathogenesis of tumors. For the treat-ment of chronic diseases, prolonged useof IFN-a may be required, as is thepractice with hormone replacement orantihypertensive therapy.

Before a therapeutic approach, such asa recombinant protein, can have wide-spread clinical application, it must en-dure a long process, starting with basicresearch, leading to development and fi-nally to arduous and hopefully creativeclinical investigation (207). With the in-creasing interaction of biochemists, ge-neticists, cell biologists, pharmacolo-gists, and clinical scientists in both theacademic community and drug develop-ment industry, the extraordinary expan-sion ofknowledge now occurring in basicscience and human diseases certainly willbe translated into benefit for countlessnumbers of people in the future.

While an attempt has been made to ac-knowledge the pivotal papers in the differentacademic fields covered within this essay, thespace limitations required the citation of onlya portion; any omissions are regretted. I amgrateful to the many colleagues who providedexplanations, comments, and unpublished re-suits for this review. Special thanks go to PollyDuff, Brenda Lethridge, and Evelyn Moorefor transcribing numerous audiotapes, CarolHunter for literature research, and Geri Park

for assistance with preparation of the manu-script. The research was conducted in part bythe Clayton Foundation and also by the Bio-medical Research Foundation.

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