MAD analysis of FHIT, a putative human tumor suppressor from the HIT protein family

BACKGROUND: The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. It is expressed from a gene located at a fragile site on human chromosome 3, which is commonly disrupted in association with certain cancers. On the basis of the genetic evidence, it has been postulated that the FHIT protein may function as a tumor suppressor, implying a role for the FHIT protein in carcinogenesis. The FHIT protein has dinucleoside polyphosphate hydrolase activity in vitro, thus suggesting that its role in vivo may involve the hydrolysis of a phosphoanhydride bond. The structural analysis of FHIT will identify critical residues involved in substrate binding and catalysis, and will provide insights into the in vivo function of HIT proteins. RESULTS: The three-dimensional crystal structures of free and nucleoside complexed FHIT have been determined from multiwavelength anomalous diffraction (MAD) data, and they represent some of the first successful structures to be measured with undulator radiation at the Advanced Photon Source. The structures of FHIT reveal that this protein exists as an intimate homodimer, which is based on a core structure observed previously in another human HIT homolog, protein kinase C interacting protein (PKCI), but has distinctive elaborations at both the N and C termini. Conserved residues within the HIT family, which are involved in the interactions of the proteins with nucleoside and phosphate groups, appear to be relevant for the catalytic activity of this protein. CONCLUSIONS: The structure of FHIT, a divergent HIT protein family member, in complex with a nucleotide analog suggests a metal-independent catalytic mechanism for the HIT family of proteins. A structural comparison of FHIT with PKCI and galactose-1-phosphate uridylyltransferase (GaIT) reveals additional implications for the structural and functional evolution of the ubiquitous HIT family of proteins.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.     

Crystallographic analysis of substrate binding and catalysis in dihydrolipoyl transacetylase (E2p)

The catalytic domain of dihydrolipoyl transacetylase (E2pCD) forms the core of the pyruvate dehydrogenase multienzyme complex and catalyzes the acetyltransferase reaction using acetylCoA as acetyl donor and dihydrolipoamide (Lip(SH)2) as acceptor. The crystal structures of six complexes and derivatives of Azotobacter vinelandii E2pCD were solved. The binary complexes of the enzyme with CoA and Lip(SH)2 were determined at 2.6- and 3.0-A resolutions, respectively. The two substrates are found in an extended conformation at the two opposite entrances of the 30 A long channel which runs at the interface between two 3-fold-related subunits and forms the catalytic center. The reactive thiol groups of both substrates are within hydrogen-bond distance from the side chain of His 610. This fact supports the indication, derived from the similarity with chloramphenicol acetyl transferase, that the histidine side chain acts as general-base catalyst in the deprotonation of the reactive thiol of CoA. The conformation of Asn 614 appears to be dependent on the protonation state of the active site histidine, whose function as base catalyst is modulated in this way. Studies on E2pCD soaked in a high concentration of dithionite lead to the structure of the binary complex between E2pCD and hydrogen sulfite solved at 2.3-A resolution. It appears that the anion is bound in the middle of the catalytic center and is therefore capable of hosting and stabilizing a negative charge, which is of special interest since the reaction catalyzed by E2pCD is thought to proceed via a negatively charged tetrahedral intermediate. The structure of the binary complex between E2pCD and hydrogen sulfite suggests that transition-state stabilization can be provided by a direct hydrogen bond between the side chain of Ser 558 and the oxy anion of the putative intermediate. In the binary complex with CoA, the hydroxyl group of Ser 558 is hydrogen bonded to the nitrogen atom of one of the two peptide-like units of the substrate. Thus, CoA itself is involved in keeping the Ser hydroxyl group in the proper position for transition-state stabilization. Quite unexpectedly, the structure at 2.6-A resolution of a ternary complex in which CoA and Lip(SH)2 are simultaneously bound to E2pCD reveals that CoA has an alternative, nonproductive binding mode. In this abortive ternary complex, CoA adopts a helical conformation with two intramolecular hydrogen bonds and the reactive sulfur of the pantetheine arm positioned 12 A away from the active site residues involved in the transferase reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence analysis of the AAA protein family

The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.     

Structure-based design of a dimeric zinc finger protein

In 1996, the dominant (43%) strain of vancomycin-resistant enterococci (VRE; type A) at Massachusetts General Hospital was identified at Brigham and Women's Hospital (BWH). To characterize the epidemiology of infection with type A isolates of VRE at BWH, we collected demographic and clinical data for all patients from whom VRE were isolated from a clinical specimen through September 1996. The first clinical isolates from all BWH patients from whom VRE were isolated were typed by pulsed-field gel electrophoresis of SmaI digests of chromosomal DNA. Among patients hospitalized after the first patient at BWH infected with a type A isolate of VRE was identified, exposures were compared between patients who acquired type A isolates of VRE and those who acquired other types of VRE. Isolates from 99 patients identified to have acquired VRE were most commonly from blood (n = 27), urine (n = 19), or wounds (n = 19). Three months after the index patient arrived at BWH and at a time when > or =12 types of strains of VRE were present, type A isolates of VRE became dominant; 39 of 75 (52%) of the study cohort had acquired type A isolates of VRE. We found no association between the acquisition of type A isolates of VRE and transfer from another institution or temporal overlap by service, ward, or floor with patients known to have acquired type A isolates of VRE. By multivariate analysis, only residence in the medical intensive care unit (adjusted odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4 to 107) and the receipt of two or more antibiotics per patient-day (adjusted OR, 12.2; 95% CI, 1.2 to 9.0) were associated with the acquisition of strain A. This strain of VRE, dominant at two Boston hospitals, was associated with intensity of antibiotic exposures (i.e., two or more antibiotics per patient-day). We hypothesize that this strain may have unidentified properties providing a mechanism favoring its spread and dominance over other extant isolates, and further studies are needed to define these properties.     

Structure-based design of ligands for protein basic domains: application to the HIV-1 Tat protein

OBJECTIVES: to describe the clinical, microbiological and pathological features of invasive sinus aspergillosis affecting immunocompetent hosts, and to identify the risk factors for mortality. METHODS: we report three apparently immunocompetent patients with invasive sinus aspergillosis, and review all cases reported in the English literature since 1987, the year in which the triazole antifungal agents were introduced. RESULTS: twenty-nine patients (including three of our own) were identified. The presenting symptoms were non-specific and indistinguishable from viral, bacterial or allergic causes of sinusitis. The findings on computed tomography scan were also non-specific, and histopathology and culture of sinus tissue biopsy had low yield. These factors, along with the mistaken impression that Aspergillus can only affect immunocompromised hosts, frequently delayed the diagnosis. Fifty-nine percent of patients either failed therapy or died. The following factors were associated with a poor prognosis: delayed diagnosis, intracranial extension of infection, and histopathology demonstrating hyphal invasion of blood vessel or tissue. Complete surgical extirpation was the key element of successful therapy; antifungal agents played an adjunctive role. CONCLUSIONS: invasive sinus aspergillosis carries high morbidity and mortality, even in immunocompetent hosts. To improve outcome, the diagnosis must be recognized early, before the organism can invade the central nervous system or vascular structures. Aggressive surgical resection of the infected areas is of utmost importance in the management of this infection.     

Kinetic analysis of the induction period in lipoxygenase catalysis

The dioxygenation of 50 microM linoleate at 0.1 microM (13S)-hydroperoxylinoleate, 240 microM O2, pH 10, and 25 degrees C, catalyzed by varying amounts of soybean lipoxygenase-1, was studied with rapid kinetic techniques. The aim was to assess the effect of transient redistributions of the Fe(II) and Fe(III) enzyme forms on the shape of the reaction progress curves. Reactions initiated with iron(II) lipoxygenase show an initial increase in rate, the "kinetic lag phase" or "induction period". The duration of this induction period varies from approximately 1 s at [lipoxygenase] > 20 nM to 5 s at [lipoxygenase] = 3 nM. At [lipoxygenase] < 2 nM, the duration of the induction period in these curves is inversely proportional to [lipoxygenase]. The integrated steady-state rate equation for the single fatty acid binding site model of lipoxygenase catalysis [Schilstra et al. (1992) Biochemistry 31, 7692-7699] also shows an induction period whose duration is inversely proportional to [lipoxygenase]. These observations, in combination with non-steady-state numerical simulations, lead to the conclusion that, at [lipoxygenase] < 2 nM, pre-steady-state redistributions of enzyme intermediates occur fast with respect to the rate at which the concentrations of substrates and products change. At higher lipoxygenase concentrations, the pre-steady-state redistributions contribute significantly to the induction period. From a nonlinear least-squares fit to the steady-state rate equation of data obtained at lipoxygenase concentrations of 0.5 and 1 nM, it was calculated that 1% of the linoleate radicals that are formed after hydrogen abstraction dissociate from the active site before enzymic oxygen insertion has occurred.     

Structure-based inhibitor design by using protein models for the development of antiparasitic agents

The lack of an experimentally determined structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm experimentally the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-1-naphthoic acid, Ki = 3 microM] and malaria (oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 microM) parasite life cycles.     

Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase

Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP.     

Shear numbers of protein beta-barrels: definition refinements and statistics

The original definition of shear number for a beta-barrel is not unique if it contains one or more uneven beta-bulges. We define the shear number of a beta-barrel as the minimal change of residue numbers in the backbone direction for all closed paths on the beta-barrel. We also discuss how to overcome some computational difficulties. It is pointed out that some closed beta-sheets should not be considered as beta-barrels. The pertinent statistics obtained from a representative list of the Protein Data Bank entries are summarized. All beta-barrels have positive shear numbers, i.e. they are right-twisted. The shear numbers of most beta-barrels are even, but exceptions do exist. The sizes of beta-ladders in a beta-barrel vary significantly. Most beta-barrels contain uneven beta-bulges, which may have important biological functions.     

Role of water in protein kinase C catalysis and its binding to membranes

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.     

Homoserine dehydrogenase-I (Escherichia coli): action of monovalent ions on catalysis and substrate association-dissociation

Changes in the kinetic properties of homoserine dehydrogenase-I (HD-I) from Escherichia coli, caused by substitution of Na+ for the normal activating monovalent ion, K+, has been investigated by equilibrium isotope exchange kinetics (EIEK). HD-I, part of the aspartokinase/homoserine dehydrogenase-I complex, is one of the few dehydrogenases to exhibit allosteric feedback regulation and cation activation. EIEK methods are especially useful for definitively identifying which rate constants are altered by bound modifiers. Saturation curves for the [14C]Hse<-->ASA and [3H]NADP+<-->NADPH exchanges were compared in the presence of K+ vs Na+, varying different combinations of substrate pairs in constant ratio at equilibrium. Kinetic differences between the K+ and Na+ enzymes were analyzed systematically by simulations with the ISOBI program. This analysis clearly demonstrates that substituting Na+ for K+ shifts the kinetic mechanism from preferred order random to a nearly random order scheme, along with causing significant rate limitation at catalysis between the central complexes. Initial velocity kinetics demonstrate that HD-I has a 10-fold higher affinity for Na+ than K+, but that the Na(+)-enzyme is 10-fold less active and exhibits higher substrate Km values, especially for L-Hse.     

Measured change in protein solvation with substrate binding and turnover

Osmotic stress is used to measure solvation changes that accompany the conformational changes of an active enzyme. For hexokinase both the equilibrium dissociation constant and the kinetic Michaelis-Menten constant for glucose vary linearly, and to the same extent, with the activity of water in the protein medium, as adjusted with large molecular weight (> 2000) osmolytes. The variation over the whole osmotic pressure range studied indicates that glucose binding is accompanied by the release of at least 65 +/- 10 water molecules, and this is reversed on enzyme turnover. The results indicate that near the physiological range of pressures the number may be higher. Most of this water, which behaves like an inhibitor, likely comes from the cleft which is induced to close around the substrate. Such large dehydration/rehydration reactions during turnover imply a significant contribution of solvation to the energetics of the conformational changes. Osmotic stress is a method of general applicability to probe water's contribution to functioning molecules.     

Precision substrate targeting of protein kinases v-Abl and c-Src

The active site substrate specificities of v-Abl and c-Src are compared and contrasted. Both enzymes catalyze the phosphorylation of a broad assortment of peptide-bound aliphatic and aromatic alcohols, such as achiral and simple straight chain residues. In addition, both protein kinases exhibit a "dual specificity" with respect to the ability to utilize D- and L-configurational isomers as substrates. However, c-Src and v-Abl are extremely inefficient as catalysts for certain structural arrangements, including secondary alcohols and primary alcohols containing large substituents in close proximity to the hydroxyl moiety. In addition to these similarities, these enzymes also display noteworthy differences in catalytic behavior. Whereas c-Src exhibits a modest preference for aromatic versus aliphatic alcohols, v-Abl does not. Most dramatic is the ability of c-Src to utilize short chain alcohols as substrates, an activity virtually absent from the catalytic repertoire of v-Abl. The implications of these observations are 2-fold. First, because both enzymes are able to accommodate a wide variety of structural variants within their respective active site regions, there exists a substantial degree of flexibility with respect to inhibitor design. Second, because these enzymes exhibit disparate active site specificities, it is possible that other tyrosine-specific protein kinases will display unique substrate specificities as well. Consequently, it may ultimately be possible to exploit these differences to generate inhibitors that precisely target specific protein kinases.     

Comments on the analysis of substrate cracking and spalling experiments

Data on crack spalling and on residual stress induced substrate cracking are reanalysed using a global energy release rate partitioning scheme. The stability of the crack path is analysed in terms of the maxima in the mode I component. This is compared with the previous analysis based on local stress field partitioning. It is suggested that some microcracking may have occurred and an attempt at including friction effects is given. It is concluded that crucial experiments would require very large substrate depths.     

Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis

The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.     

Structural and transglutaminase substrate properties of the small proline-rich 2 family of cornified cell envelope proteins

The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no alpha or beta structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substrate in vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2.     

Breast cancer and family history: a multivariate analysis of levels of tumor HER2 protein and family history of cancer in women who have breast cancer

BACKGROUND: The HER2 gene, located on the long arm of chromosome 17, codes for a protein with the characteristics of a growth factor receptor. In a preliminary study, we reported that high levels of tumor HER2 (erbB-2/neu) protein are associated with a family history of breast cancer (that is, one or more female blood relatives with breast cancer). METHODS: We have now collected a larger number of subjects (94) and performed a multivariate analysis of the independent variables family history of breast cancer, tumor estrogen receptor, age, and tumor DNA index. Family history of breast cancer was assessed by questioning the patient, in many cases by telephone. RESULTS: HER2 levels were significantly higher in women with a family history of breast cancer (p = 0.015, two-tailed t-test). The 27 women with family history were predominantly postmenopausal, mean age 61 +/- 2.3 (mean +/- SEM), versus a mean age of 56 +/- 1.7 for the 67 women with no family history. Of the 27 women with a family history of breast cancer, 13 had a first-degree relative (mother or sister) with the disease. The remaining 14 women had other relatives (grandmothers, aunts, cousins, or a niece) with breast cancer. The results of multiple linear regression analysis, with HER2 as the dependent variable, showed that family history of breast cancer was significantly associated with elevated HER2 levels in the tumors (p = 0.0038), after controlling for the effects of age, tumor estrogen receptor, and DNA index. CONCLUSIONS: The association of family history of breast cancer and elevated tumor HER2 protein suggests that postmenopausal familial breast cancer may be associated with altered HER2 expression.     

Bone morphogenetic protein and bone morphogenetic protein gene family in bone formation and repair

The bone morphogenetic proteins are secreted signalling molecules that belong to the transforming growth factor beta family of growth and differentiation factors. Individual bone morphogenetic proteins are prominent at many sites during embryogenesis and are likely to be key regulators of early development and organogenesis. In vertebrates, one of the functions of bone morphogenetic like proteins is to induce formation of bone, cartilage, and connective tissues associated with the skeleton. This osteoinductive ability has led to the use of bone morphogenetic proteins as therapeutic agents for creation of new bone useful in treatment of skeletal injuries and diseases, and in oral and maxillofacial applications.     

Serological analysis of human tumor antigens: molecular definition and implications

Specific vaccines for the immunotherapy of human neoplasms require specific human tumor antigens. While efforts to identify such antigens by the analysis of the T-cell repertoire have yielded few antigens, the application of SEREX, the serological identification of antigens by recombinant expression cloning, has brought a cornucopia of new antigens. Several specific antigens have been identified in each tumor tested, suggesting that many human tumors elicit multiple immune responses in the autologous host. The frequency of human tumor antigens, which can be readily defined at the molecular level, facilitates the identification of T-cell-dependent antigens and provides a basis for peptide and gene-therapeutic vaccine strategies.