Structural basis for activation of Arf1 at the Golgi complex.

The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.

Mechanisms of SSBP1 variants in mitochondrial disease: Molecular dynamics simulations reveal stable tetramers with altered DNA binding surfaces.

Several mutations in the gene for the mitochondrial single stranded DNA binding protein (SSBP1) have recently been implicated in human disease, but initial reports are insufficient to explain the molecular mechanism of disease, including the possible role of SSBP1 heterotetramers in heterozygous patients. Here we employed molecular simulations to model the dynamics of wild type and 31 variant SSBP1 tetramer systems, including 7 variant homotetramer and 24 representative heterotetramer systems. Our simulations indicate that all variants are stable and most have stronger intermonomer interactions, reduced solvent accessible surface areas, and a net loss of positive surface charge. We then used structural alignments and phosphate binding simulations to predict DNA binding surfaces on SSBP1. Our models suggest that nearly the entire surface of SSBP1, excluding flexible loops and protruding helices, is available for DNA binding, and we observed several potential DNA binding hotspots. Changes to the protein surface in variant SSBP1 tetramers potentially alter anchor points or wrapping paths, rather than abolishing binding altogether. Overall, our findings disqualify tetramer destabilization or gross disruption of DNA binding as mechanisms of disease. Instead, they are consistent with subtle changes to DNA binding, wrapping, or release that cause rare but consequential failures of mtDNA maintenance, which, in turn, are consistent with the late onset of disease in most of the reported SSBP1 cases.

Consequences of compromised mitochondrial genome integrity.

Maintenance and replication of the mitochondrial genome (mtDNA) is essential to mitochondrial function and eukaryotic energy production through the electron transport chain. mtDNA is replicated by a core set of proteins: Pol γ, Twinkle, and the single-stranded DNA binding protein. Fewer pathways exist for repair of mtDNA than nuclear DNA, and unrepaired damage to mtDNA may accumulate and lead to dysfunctional mitochondria. The mitochondrial genome is susceptible to damage by both endogenous and exogenous sources. Missense mutations to the nuclear genes encoding the core mtDNA replisome (POLG, POLG2, TWNK, and SSBP1) cause changes to the biochemical functions of their protein products. These protein variants can damage mtDNA and perturb oxidative phosphorylation. Ultimately, these mutations cause a diverse set of diseases that can affect virtually every system in the body. Here, we briefly review the mechanisms of mtDNA damage and the clinical consequences of disease variants of the core mtDNA replisome.

SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder.

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.

Mitochondrial single-stranded DNA binding protein novel de novo SSBP1 mutation in a child with single large-scale mtDNA deletion (SLSMD) clinically manifesting as Pearson, Kearns-Sayre, and Leigh syndromes.

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband's fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication.

Regulation of Arf activation occurs via distinct mechanisms at early and late Golgi compartments.

At the Golgi complex, the biosynthetic sorting center of the cell, the Arf GTPases are responsible for coordinating vesicle formation. The Arf-GEFs activate Arf GTPases and are therefore the key molecular decision-makers for trafficking from the Golgi. In Saccharomyces cerevisiae, three conserved Arf-GEFs function at the Golgi: Sec7, Gea1, and Gea2. Our group has described the regulation of Sec7, the trans-Golgi Arf-GEF, through autoinhibition, positive feedback, dimerization, and interactions with a suite of small GTPases. However, we lack a clear understanding of the regulation of the early Golgi Arf-GEFs Gea1 and Gea2. Here we demonstrate that Gea1 and Gea2 prefer neutral over anionic membrane surfaces in vitro, consistent with their localization to the early Golgi. We illustrate a requirement for a critical mass of either Gea1 or Gea2 for cell growth under stress conditions. We show that the C-terminal domains of Gea1 and Gea2 toggle roles in the cytosol and at the membrane surface, preventing membrane binding in the absence of a recruiting interaction but promoting maximum catalytic activity once recruited. We also identify the small GTPase Ypt1 as a recruiter for Gea1 and Gea2. Our findings illuminate core regulatory mechanisms unique to the early Golgi Arf-GEFs.

The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase.

The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

Developing continuous variable composites for Rorschach measures of thought problems, vigilance, and suicide risk.

Using a multiple regression approach with a large developmental sample (N = 460) of Rorschach protocols from psychiatric, forensic, and nonclinical control groups, the authors created continuous multivariable Composite scores corresponding to the Comprehensive System (CS) Perceptual-Thinking Index, Hypervigilance Index, and Suicide Constellation. Within a validation sample (N = 230), these three new scores, called the Thought and Perception Composite, Vigilance Composite, and Suicide Concern Composite were strongly associated with the three original CS Indices. Additional analyses suggest that the new Composite scores were more reliable than and at least as valid as the original Indices. Interpretive guidelines are offered.

In vivo repair of alkylating and oxidative DNA damage in the mitochondrial and nuclear genomes of wild-type and glycosylase-deficient Caenorhabditis elegans.

Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is critical for repair of many of the most common types of DNA damage generated both by endogenous metabolic pathways and exposure to exogenous stressors such as pollutants. Caenorhabditis elegans is an increasingly important model organism for the study of DNA damage-related processes including DNA repair, genotoxicity, and apoptosis, but BER is not well understood in this organism, and has not previously been measured in vivo. We report robust BER in the nuclear genome and slightly slower damage removal from the mitochondrial genome; in both cases the removal rates are comparable to those observed in mammals. However we could detect no deficiency in BER in the nth-1 strain, which carries a deletion in the only glycosylase yet described in C. elegans that repairs oxidative DNA damage. We also failed to detect increased lethality or growth inhibition in nth-1 nematodes after exposure to oxidative or alkylating damage, suggesting the existence of at least one additional as-yet undetected glycosylase.

Human mitochondrial DNA polymerase γ exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers.

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3'-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3'-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.

Importance of the C2, N7, and C8 positions to the mutagenic potential of 8-Oxo-2'-deoxyguanosine with two A family polymerases.

8-Oxo-2'-deoxyguanosine (OdG) is a prominent DNA lesion produced from the reaction of 2'-deoxyguanosine (dG) with reactive oxygen species. While dG directs the insertion of only dCTP during replication, OdG can direct the insertion of either dCTP or dATP, allowing for the production of dG → dT transversions. When replicated by Klenow fragment-exo (KF-exo), OdG preferentially directs the incorporation of dCTP over dATP, thus decreasing its mutagenic potential. However, when replicated by a highly related polymerase, the large fragment of polymerase I from Bacillus stearothermophilus (BF), dATP incorporation is preferred, and a higher mutagenic potential results. To gain insight into the reasons for this opposite preference and the effects of the C2, N7, and C8 positions on OdG mutagenicity, single-nucleotide insertions of dCTP and/or dATP opposite dG, OdG, and seven of their analogues were examined by steady state kinetics with both KF-exo and BF. Results from these studies suggest that the two enzymes behave similarly and are both sensitive not only to steric and electronic changes within the imidazole ring during both dCTP and dATP incorporation but also to the presence of the C2-exocyclic amine during dATP incorporation. The difference in incorporation preference opposite OdG appears to be due to a somewhat increased sensitivity to structural perturbations during dCTP incorporation with BF. Single-nucleotide extensions past the resulting base pairs were also studied and were not only similar between the two enzymes but also consistent with published ternary crystallographic studies with BF. These results are analyzed in the context of previous biochemical and structural studies, as well as stability studies with the resulting base pairs.

Nuclear imaging of Met-expressing human and canine cancer xenografts with radiolabeled monoclonal antibodies (MetSeek).

PURPOSE: Met, an oncogene product and receptor tyrosine kinase, is a keystone molecule for malignant progression in solid human tumors. We are developing Met-directed imaging and therapeutic agents, including anti-Met monoclonal antibodies (MetSeek). In this study, we compared two antibodies, Met5 and Met3, for nuclear imaging of human and canine Met-expressing tumor xenografts in nude mice. EXPERIMENTAL DESIGN: Xenografts representing cancers of three different human tissue origins and metastatic canine prostate cancer were raised s.c. in host athymic nude mice. Animals were injected i.v. with I-125-Met5 or I-125-Met3, posterior total body gamma camera images were acquired for several days postinjection, and quantitative region-of-interest activity analysis was done. RESULTS: PC-3, SK-LMS-1/HGF, and CNE-2 xenografts imaged with I-125-Met5 were compared with PC-3, SK-LMS-1/HGF, and DU145 xenografts imaged with I-125-Met3. Nuclear imaging contrast was qualitatively similar for I-125-Met5 and I-125-Met3 in PC-3 and SK-LMS-1/HGF host mice. However, by region-of-interest analysis, the set of human tumors imaged with I-125-Met3 exhibited a pattern of rapid initial tumor uptake followed by a continuous decline in activity, whereas the set of human tumors imaged with I-125-Met5 showed slow initial uptake, peak tumor-associated activity at 1 day postinjection, and persistence of activity in xenografts for at least 5 days. GN4 canine prostate cancer xenografts were readily imaged with I-125-Met5. CONCLUSIONS: We conclude that radioiodinated Met3 and Met5 offer qualitatively similar nuclear images in xenograft-bearing mice, but quantitative considerations indicate that Met5 might be more useful for radioimmunotherapy. Moreover, canine prostate cancer seems to be a suitable model for second-stage preclinical evaluation of Met5.

Proliferation and invasion: plasticity in tumor cells.

Invasive and proliferative phenotypes are fundamental components of malignant disease, yet basic questions persist about whether tumor cells can express both phenotypes simultaneously and, if so, what are their properties. Suitable in vitro models that allow characterization of cells that are purely invasive are limited because proliferation is required for cell maintenance. Here, we describe glioblastoma cells that are highly invasive in response to hepatocyte growth factor/scatter factor (HGF/SF). From this cell population, we selected subclones that were highly proliferative or displayed both invasive and proliferative phenotypes. The biological activities of invasion, migration, urokinase-type plasminogen activation, and branching morphogenesis exclusively partitioned with the highly invasive cells, whereas the highly proliferative subcloned cells uniquely displayed anchorage independent growth in soft agar and were highly tumorigenic as xenografts in immune-compromised mice. In response to HGF/SF, the highly invasive cells signal through the MAPK pathway, whereas the selection of the highly proliferative cells coselected for signaling through Myc. Moreover, in subcloned cells displaying both invasive and proliferative phenotypes, both signaling pathways are activated by HGF/SF. These results show how the mitogen-activated protein kinase and Myc pathways can cooperate to confer both invasive and proliferative phenotypes on tumor cells and provide a system for studying how transitions between invasion and proliferation can contribute to malignant progression.

Geldanamycins exquisitely inhibit HGF/SF-mediated tumor cell invasion.

Induction of the urokinase-type plasminogen activator (uPA) by hepatocyte growth factor/scatter factor (HGF/SF) plays an important role in tumor cell invasion and metastasis that is mediated through the Met receptor tyrosine kinase. Geldanamycins (GA) are antitumor drugs that bind and inhibit HSP90 chaperone activity at nanomolar concentrations (nM-GAi) by preventing proper folding and functioning of certain oncoproteins. Previously, we have shown that a subset of GA derivatives exhibit exquisite potency, inhibiting HGF/SF-induced uPA-plasmin activation at femtomolar concentrations (fM-GAi) in canine MDCK cells. Here, we report that (1) inhibition of HGF/SF-induced uPA activity by fM-GAi is not uncommon, in that several human tumor glioblastoma cell lines (DBTRG, U373 and SNB19), as well as SK-LMS-1 human leiomyosarcoma cells are also sensitive to fM-GAi; (2) fM-GAi drugs only display inhibitory activity against HGF/SF-induced uPA activity (rather than basal activity), and only when the observed magnitude of uPA activity induction by HGF/SF is at least 1.5 times basal uPA activity; and (3) not only do fM-GAi derivatives strongly inhibit uPA activity but they also block MDCK cell scattering and in vitro invasion of human glioblastoma cells at similarly low drug concentrations. These effects of fM-GAi drugs on the Met-activated signaling pathway occur at concentrations well below those required to measurably affect Met expression or cell proliferation. We also examined the effect of Radicicol (RA), a drug with higher affinity than GA for HSP90. RA displays uPA activity inhibition at nanomolar levels, but not at lower concentrations, indicating that HSP90 is not likely the fM-GAi molecular target. Thus, we show that certain GA drugs (fM-GAi) in an HGF/SF-dependent manner block uPA-plasmin activation in tumor cells at femtomolar levels. This inhibition can also be observed in scattering and in vitro invasion assays. Our findings also provide strong circumstantial evidence for a novel non-HSP90 molecular target that is involved in HGF/SF-mediated tumor cell invasion.

Enhanced growth of human met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor.

Downstream signaling that results from the interaction of hepatocyte growth factor/scatter factor (HGF/SF) with the receptor tyrosine kinase Met plays critical roles in tumor development, progression, and metastasis. This ligand-receptor pair is an attractive target for new diagnostic and therapeutic agents, preclinical development of which requires suitable animal models. The growth of heterotopic and orthotopic Met-expressing human tumor xenografts in conventional strains of immunocompromised mice inadequately replicates the paracrine stimulation by human HGF/SF (hHGF/SF) that occurs in humans with cancer. We have therefore generated a mouse strain transgenic for hHGF/SF (designated hHGF-Tg) on a severe combined immunodeficiency (SCID) background. We report here that the presence of ectopically expressed hHGF/SF ligand significantly enhances growth of heterotopic subcutaneous xenografts derived from human Met-expressing cancer cells, including the lines SK-LMS-1 (human leiomyosarcoma), U118 (human glioblastoma), and DU145 (human prostate carcinoma), but not that of M14-Mel xenografts (human melanoma that expresses insignificant levels of Met). Our results indicate that ectopic hHGF/SF can specifically activate Met in human tumor xenografts. This new hHGF-Tg strain of mice should provide a powerful tool for evaluating drugs and diagnostic agents that target the various pathways influenced by Met activity.

RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival.

Hepatocyte growth factor/scatter factor-Met signaling has been implicated in tumor growth, invasion, and metastasis. Suppression of this signaling pathway by targeting the Met protein tyrosine kinase may be an ideal strategy for suppressing malignant tumor growth. Using RNA interference technology and adenovirus vectors carrying small-interfering RNA constructs (Ad Met small-interfering RNA) directed against mouse, canine, and human Met, we can knock down c-met mRNA. We show a dramatic dependence on Met in both ligand-dependent and ligand-independent mouse, canine, and human tumor cell lines. Mouse mammary tumor (DA3) cells and Met-transformed NIH3T3 (M114) cells, as well as both human and canine prostate cancer (PC-3 and TR6LM, human sarcoma (SK-LMS-1), glioblastoma (DBTRG), and gastric cancer (MKN45) cells, all display a dramatic reduction of Met expression after infection with Ad Met small-interfering RNA. In these cells, we observe suppression of tumor cell growth and viability in vitro as well as inhibition of hepatocyte growth factor/scatter factor-mediated scattering and invasion in vitro, whether Met activation was ligand dependent or not. Importantly, Ad Met small-interfering RNA led to apoptotic cell death in many of the tumor cell lines, especially DA3 and MKN45, but did not adversely affect MDCK canine kidney cells. Met small-interfering RNA also abrogated downstream Met signaling to molecules such as Akt and p44/42 mitogen-activated protein kinase. We further show that intratumoral infection with c-met small-interfering RNA adenovirus results in a substantial reduction in tumor growth. Thus, Met small-interfering RNA adenoviruses are reliable tools for studying Met function and raise the possibility of their application for cancer therapy.

Overexpression of sprouty 2 inhibits HGF/SF-mediated cell growth, invasion, migration, and cytokinesis.

A strict regulation of hepatocyte growth factor/scatter factor (HGF/SF)-Met signaling is essential for its appropriate function. Several negative regulators of Met signaling have been identified. Here we report that human Spry2 is induced by HGF/SF and negatively regulates HGF/SF-Met signaling. We show that overexpression of Spry2 inhibits cell proliferation, anchorage-independent cell growth, and migration in wound-healing and in vitro invasion assays. Measured in an electric cell-substrate impedance sensing biosensor, cell movement is restricted, because Spry2 dramatically facilitates cell attachment and spreading by enhancing focal adhesions and increasing stress fibers. An analysis of cell cycle distribution shows, unexpectedly, that Spry2-GFP cells are polyploid. Thus, as with FGF and EGF receptors, Spry2-GFP tempers downstream Met signaling in addition to its pronounced effect on cell adhesion, and it has properties suitable to be considered a tumor-suppressor protein.

Radioimmunoscintigraphy of human met-expressing tumor xenografts using met3, a new monoclonal antibody.

PURPOSE: Inappropriate expression of the receptor tyrosine kinase Met and its ligand is associated with an aggressive phenotype and poor clinical prognosis for a wide variety of solid human tumors. We are developing imaging and therapeutic agents that target this receptor-ligand complex. In this study, we evaluated the ability of radioiodinated anti-Met monoclonal antibodies from a single hybridoma clone to image human Met-expressing tumor xenografts. EXPERIMENTAL DESIGN: Xenografts of four different tissue origins were raised s.c. in host athymic nude mice. Animals received i.v. injections of I-125-Met3, posterior total body gamma camera images were acquired for several days after injection, and quantitative region-of-interest activity analysis was performed. RESULTS: The autocrine Met-expressing tumors S-114 and SK-LMS-1/HGF and the paracrine Met-expressing human prostate carcinoma PC-3 were satisfactorily imaged with I-125-Met3. By region-of-interest analysis, mean initial tumor-associated activities in S-114, SK-LMS-1/HGF, and PC-3 were 18.6 +/- 2.1, 7.2 +/- 2.2, and 5.4 +/- 2.6% estimated injected activity, and the mean ratios of tumor:total body activity at 3 days after injection were 0.32 +/- 0.13, 0.15 +/- 0.06, and 0.10 +/- 0.04, respectively. Human melanoma xenografts, however, accounted for < or =3% of injected or total body activity. We observed a direct rank order correlation between relative levels of Met3-derived radioactivity in xenografts and relative quantities of Met expressed by the respective cultured tumor cell lines. CONCLUSIONS: We conclude that I-125-Met3 is effective for imaging human Met-expressing xenografts of different tissue origins, and we infer that I-125-Met3 distinguishes human tumor xenografts according to their levels of Met expression.