Neurologic complications after allogeneic hematopoietic stem cell transplantation: risk factors and impact.

Neurologic complications (NCs) may be a significant source of morbidity and mortality after hematopoietic cell transplantation (HCT). We performed a retrospective study of 263 consecutive patients undergoing allogeneic HCT for hematological malignancies to determine the incidence, risk factors and clinical impact of NCs in the first 5 years after HCT. We determined the incidence of central nervous system (CNS) infection, intracranial hemorrhage, ischemic stroke, metabolic encephalopathy, posterior reversal encephalopathy syndrome, seizure and peripheral neuropathy. In all, 50 patients experienced 63 NCs-37 early (⩽day +100), 21 late (day +101 to 2 years) and 5 very late (2 to 5 years). The 1- and 5-year cumulative incidences of all NCs were 15.6% and 19.2%, respectively, and of CNS complication (CNSC; all of the above complications except peripheral neuropathy) were 12.2 and 14.5%. Risk factors for CNSC were age (hazard ratio (HR)=1.06 per year, P=0.0034), development of acute GvHD grade III-IV (HR=2.78, P=0.041), transfusion-dependent thrombocytopenia (HR=3.07, P=0.025) and delayed platelet engraftment (>90th centile; HR=2.77, P=0.043). CNSCs negatively impacted progression-free survival (HR=2.29, P=0.0001), overall survival (HR=2.63, P<0.0001) and non-relapse mortality (HR=8.51, P<0.0001). NCs after HCT are associated with poor outcomes, and usually occur early after HCT.

Hematopoietic stem cell transplant-associated thrombotic microangiopathy: current paradigm and novel therapies.

Hematopoietic stem cell transplantation-associated thrombotic microangiopathy (TA-TMA) remains a difficult complication to address due to its high mortality rate, lack of standard diagnostic criteria and limited therapeutic options. Underscoring this challenge is the complex pathophysiology involved and multiple contributing factors that converge on a final pathway involving widespread endothelial injury and complement activation. In addressing our current understanding of TA-TMA, we highlight the risk factors leading to endothelial damage and a pathophysiological cascade that ensues. We have also compared the different definition criteria and biomarkers that can enable early intervention in TA-TMA patients. Current first-line management includes discontinuation or alteration of the immunosuppressive regimen, treatment of co-existing infectious and GVHD, aggressive hypertension control and supportive therapy. We discuss current pharmacological therapies, including newer agents that target the complement cascade and nitric oxide pathways.

Challenges to cervical cancer treatment in Bangladesh: The development of a women's cancer ward at Dhaka Medical College Hospital.

Cervical cancer is the second most common cause of female cancer mortality worldwide. Concurrent chemoradiotherapy represents the standard of care for patients with stages IB2 to IVa cervical cancer. Unfortunately radiation therapy capacity is severely limited to non-existent in many Low and Middle-Income Countries. One solution has been to use chemotherapy to reduce tumor size to allow for radical surgery or in the case of inoperable cancers, as a placeholder until radiation is available. In Bangladesh, there has been the progressive development of resources for the treatment of women with gynecologic cancers. However, radiation therapy resources are limited with a six-month waiting period to receive radiation. Neoadjuvant chemotherapy (NACT) remains the main primary treatment intervention for women with advanced cervical cancer in Bangladesh. This implementation study summarizes of the experience and challenges to caring for women in a new gynae-oncology ward at Dhaka Medical College Hospital, a 2600 bed government hospital in Dhaka, Bangladesh.

Expression of α4ß7 integrin on memory CD8(+) T cells at the presentation of acute intestinal GVHD.

Acute intestinal GVHD remains a major source of morbidity after allogeneic hematopoietic cell transplantation (HCT). α4ß7 integrin is a cell surface molecule that mediates lymphocyte trafficking to intestinal tissue. In this analysis, peripheral blood was collected at the time of presentation of symptoms of acute GVHD and before any treatment. In all, 45 samples were collected and divided into three groups on the basis of subsequent evaluation: intestinal GVHD (n=15), skin GVHD (n=20) and no GVHD (n=10). Two patients developed intestinal GVHD after DLI. The no-GVHD group comprised 10 patients who presented with suspicious symptoms, but evaluation yielded other etiologies. Analysis by flow cytometry showed that intestinal GVHD patients had a significantly higher percentage of α4ß7 integrin-expressing memory CD8(+) T cells (median 7.69%; lower and upper quartiles, 1.06% and 11.64%, respectively) compared with patients with skin GVHD (1.26%; 0.57% and 2.49%) and no GVHD (0.96%; 0.44% and 1.85%), P=0.03. No differences were found in α4ß7 expression in any CD4(+) T-cell subsets or naive CD8(+) T cells. This study adds to the evidence that α4ß7 integrin is involved in lymphocyte trafficking in acute intestinal GVHD.

Mechanisms of donor-specific tolerance in recipients of haploidentical combined bone marrow/kidney transplantation.

We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

Double umbilical cord blood transplantation with reduced intensity conditioning and sirolimus-based GVHD prophylaxis.

The main limitations to umbilical cord blood (UCB) transplantation (UCBT) in adults are delayed engraftment, poor immunological reconstitution and high rates of non-relapse mortality (NRM). Double UCBT (DUCBT) has been used to circumvent the issue of low cell dose, but acute GVHD remains a significant problem. We describe our experience in 32 subjects, who underwent DUCBT after reduced-intensity conditioning with fludarabine/melphalan/antithymocyte globulin and who received sirolimus and tacrolimus to prevent acute GVHD. Engraftment of neutrophils occurred in all patients at a median of 21 days, and platelet engraftment occurred at a median of 42 days. Three subjects had grade II-IV acute GVHD (9.4%) and chronic GVHD occurred in four subjects (cumulative incidence 12.5%). No deaths were caused by GVHD and NRM at 100 days was 12.5%. At 2 years, NRM, PFS and OS were 34.4, 31.2 and 53.1%, respectively. As expected, immunologic reconstitution was slow, but PFS and OS were associated with reconstitution of CD4(+) and CD8(+) lymphocyte subsets, suggesting that recovery of adaptive immunity is required for the prevention of infection and relapse after transplantation. In summary, sirolimus and tacrolimus provide excellent GVHD prophylaxis in DUCBT, and this regimen is associated with low NRM after DUCBT.

B-cell immunity in the context of T-cell tolerance after combined kidney and bone marrow transplantation in humans.

Five patients with end-stage kidney disease received combined kidney and bone marrow transplants from HLA haploidentical donors following nonmyeloablative conditioning to induce renal allograft tolerance. Immunosuppressive therapy was successfully discontinued in four patients with subsequent follow-up of 3 to more than 6 years. This allograft acceptance was accompanied by specific T-cell unresponsiveness to donor antigens. However, two of these four patients showed evidence of de novo antibodies reactive to donor antigens between 1 and 2 years posttransplant. These humoral responses were characterized by the presence of donor HLA-specific antibodies in the serum with or without the deposition of the complement molecule C4d in the graft. Immunofluorescence staining, ELISA assays and antibody profiling using protein microarrays demonstrated the co-development of auto- and alloantibodies in these two patients. These responses were preceded by elevated serum BAFF levels and coincided with B-cell reconstitution as revealed by a high frequency of transitional B cells in the periphery. To date, these B cell responses have not been associated with evidence of humoral rejection and their clinical significance is still unclear. Overall, our findings showed the development of B-cell allo- and autoimmunity in patients with T-cell tolerance to the donor graft.

Clinical outcomes of late rather than early full-donor chimerism in patients with advanced lymphomas receiving nonmyeloablative allogeneic hematopoietic SCT.

Allogeneic hematopoietic SCT (HSCT) can ideally provide long-term remission in advanced lymphoma patients by capturing a graft-vs-tumor (GVT) effect. On the basis of a murine model, we attempted to optimize a GVT effect through nonmyeloablative therapy and HLA-matched related donor HSCT with intentional induction of mixed chimerism followed by prophylactic donor lymphocyte infusion. A total of 26 advanced lymphoma patients were separated into an early and late full-donor chimerism (FDC) group using a median of 45 days post-HSCT as the defining point for FDC. Upon generating these groups, analysis by Student's t-test demonstrated that they were statistically distinct in time to develop FDC (P<0.01). There was a trend toward improved CR rates in the late group relative to the early group (62 vs 31%; P=0.12). A trend toward improved progression-free survival at 5 years was also observed in the late compared to the early group by Kaplan-Meier analysis (38 vs 8%; P=0.081). However, this did not correlate to a significant overall survival benefit. In conclusion, these data support the observation from our mouse models that the most potent GVT effect occurs in mixed chimeras with late chimerism conversion.

Preinfusion variables predict the predominant unit in the setting of reduced-intensity double cord blood transplantation.

Double cord blood transplantation (DCBT) may overcome the slow hematopoietic recovery and engraftment failure associated with infusion of a single cord blood unit. In DCBT, only one unit typically contributes to long-term hematopoiesis, but little is known about factors affecting cord predominance. As results from a phase I trial suggested that order of infusion may affect cord predominance, we analyzed the effect of preinfusion variables on chimerism patterns of 38 patients enrolled in the initial study and a subsequent phase II trial. All patients were treated with a reduced-intensity conditioning (RIC) regimen of fludarabine, melphalan and thymoglobulin followed by DCBT. By day 100, 66% of patients had hematopoiesis derived from a single cord blood unit. Higher post-thaw total nucleated cell and CD34+ cell dose were associated with cord predominance and in 68% of patients (P=0.03); the predominant cord blood unit was infused first. Only the post-thaw CD34+ cell dose of the predominant unit predicted time to both neutrophil and platelet engraftment. Although based on a small number of patients, our results identify parameters that may affect cord predominance and engraftment in the setting of DCBT following RIC and suggest possible strategies for selecting infusion order for cord blood units.

Comparison of outcomes after transplantation of peripheral blood stem cells versus bone marrow following an identical nonmyeloablative conditioning regimen.

This is the first study to examine the outcomes in 54 patients with hematologic malignancies who received an HLA-matched related donor bone marrow (BM, n = 42) or GCSF-mobilized peripheral blood stem cells (PBSC, n = 12) following identical nonmyeloablative conditioning with the intention of induction of mixed chimerism (MC) followed by prophylactic donor leukocyte infusion (pDLI) to convert MC to full donor chimerism (FDC) and capture a graft-versus-tumor effect without clinical graft-versus-host disease (GVHD). Neutrophil and platelet recovery were faster and transfusion requirement was less in PBSC recipients (P < 0.05). A total of 48% of BMT recipients achieved FDC with a median conversion time of 84 days, including 13 following pDLI. In contrast, 83% (P = 0.04) in the PBSC group had spontaneous FDC at a median of 14 days, precluding the administration of pDLI. There was no significant difference in the incidences of acute or chronic GVHD, though the rates of chronic GVHD were considerably higher in PBSC group than in the BM group (6/7, 86% vs 10/24, 42%). CD4 and CD8 T-cell recovery was faster in PBSC recipients. In PBSC recipients, a higher number of CD34+ cells was associated with increased rates of severe, grade III-IV acute GVHD.

Myeloma responses and tolerance following combined kidney and nonmyeloablative marrow transplantation: in vivo and in vitro analyses.

Six patients with renal failure due to multiple myeloma (MM) received simultaneous kidney and bone marrow transplantation (BMT) from HLA-identical sibling donors following nonmyeloablative conditioning, including cyclophosphamide (CP), peritransplant antithymocyte globulin and thymic irradiation. Cyclosporine (CyA) was given for approximately 2 months posttransplant, followed by donor leukocyte infusions. All six patients accepted their kidney grafts long-term. Three patients lost detectable chimerism but accepted their kidney grafts off immunosuppression for 1.3 to >7 years. One such patient had strong antidonor cytotoxic T lymphocyte (CTL) responses in association with marrow rejection. Two patients achieved full donor chimerism, but resumed immunosuppression to treat graft-versus-host disease. Only one patient experienced rejection following CyA withdrawal. He responded to immunosuppression, which was later successfully withdrawn. The rejection episode was associated with antidonor Th reactivity. Patients showed CTL unresponsiveness to cultured donor renal tubular epithelial cells. Initially recovering T cells were memory cells and were enriched for CD4+CD25+ cells. Three patients are in sustained complete remissions of MM, despite loss of chimerism in two. Combined kidney/BMT with nonmyeloablative conditioning can achieve renal allograft tolerance and excellent myeloma responses, even in the presence of donor marrow rejection and antidonor alloresponses in vitro.

Anchorage-independent growth of fibroblasts that express a truncated IGF-I receptor.

The purpose of this investigation was to study signaling by an insulin-like growth factor I receptor (IGF-I R) that lacks the extracellular portion of the receptor. We transfected IGF-I R-negative mouse embryo fibroblasts with a truncated IGF-I R consisting of only the transmembrane and cytoplasmic part of the beta subunit. Proliferation as assessed by counting cells was the same for vector only transfectants and the truncated receptor transfectants in defined medium containing EGF and PDGF. In contrast, anchorage-independent growth as measured by colony formation in soft agar was markedly increased for the truncated IGF-I R transfectants compared to the vector transfectants. MAP-kinase activity in the truncated IGF-I R transfectants was not higher than in the vector transfectants; however, PI 3-kinase activity was significantly higher in the IGF-I R transfectants. These results provide evidence that an IGF-I receptor consisting of only the transmembrane and cytoplasmic domain of the beta subunit can signal pathways leading to anchorage-independent growth.

Successful allogeneic stem cell transplantation with nonmyeloablative conditioning in patients with relapsed hematologic malignancy following autologous stem cell transplantation.

The use of myeloablative preparative therapy and allogeneic stem cell transplantation (alloSCT) as salvage therapy for adult patients with relapsed hematologic malignancy after autologous stem cell transplantation (autoSCT) is generally unsuccessful due to very high treatment-related mortality rates. We evaluated the outcome of HLA-matched related donor alloSCT following nonmyeloablative preparative therapy in 13 patients (median age, 38 years) with relapsed hematologic malignancies (Hodgkin's disease, n = 4; Hodgkin's disease and advanced myelodysplastic syndrome, n = 1; non-Hodgkin's lymphoma, n = 6; multiple myeloma, n = 2) after initial autoSCT. Median time from autoSCT to alloSCT was 12 months (range, 3-24 months); 6 patients had chemotherapy-refractory disease following autoSCT, 6 were in untreated relapse, and 1 had a partial response from salvage chemotherapy. Preparative therapy consisted of cyclophosphamide, 150-200 mg/kg; peritransplantation anti-thymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal irradiation); and a very short course of cyclosporine as GVHD prophylaxis. All patients achieved initial mixed chimerism as defined by greater than 1% donor peripheral white blood cells. Seven patients, who had no evidence of GVHD, received prophylactic DLI beginning 5 to 6 weeks after transplantation for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-tumor effect. Six patients showed conversion to full donor chimerism and 1 lost the graft. Grade II or greater acute GVHD occurred in 9 patients. Seven patients achieved a complete response; 6 had no response. The median survival time of the 13 patients is currently 10 months (range, 3-39 months), with an overall survival probability at 2 years of 45% (95% confidence interval [CI], 19%-73%) and a disease-free survival probability at 2 years of 37.5% (95% CI, 12%-65%). Thus, this novel nonmyeloablative alloSCT strategy followed by prophylactic DLI was well tolerated and can result in durable disease-free survival among patients with advanced hematologic malignancies after a failed autoSCT. Further follow-up and evaluation of additional patients are required to conclusively establish the role of this strategy in the treatment of hematologic malignancies after an autologous transplantation.

Suppressor of cytokine signaling (SOCS)-3 protein interacts with the insulin-like growth factor-I receptor.

SOCS proteins are a class of proteins that are negative regulators of cytokine receptor signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. In a yeast two-hybrid screen of a human fetal brain library, we have previously identified SOCS-2 as a binding partner of the activated IGF-I receptor (IGFIR). To test whether or not SOCS-3 also binds to the IGFIR, we cloned human SOCS-3 by reverse transcription-polymerase chain reaction from human skeletal muscle mRNA. SOCS-3 mRNA was expressed in many human fetal and adult tissues and in some human cancer cell lines (Hela, A549 pulmonary adenocarcinoma and G361 human melanoma). We found that human SOCS-3 protein interacts directly with the cytoplasmic domains of the activated IGFIR and the insulin receptor (IR) in the yeast two-hybrid assay. In GST-SOCS-3 pull-down experiments using IGFIR from mammalian cells and in immunoprecipitation experiments in which IGFIR and FLAG-SOCS-3 were transiently expressed in human embryonic kidney 293 cells, we found that SOCS-3 interacts constitutively with IGFIR in vitro and in intact cells. Unlike SOCS-2, hSOCS-3 was phosphorylated on tyrosines in response to IGF-I addition to 293 cells. We conclude that SOCS-3 binds to the IGFIR and may be a direct substrate for the receptor tyrosine kinase.

Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12.

We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.

Interaction of human suppressor of cytokine signaling (SOCS)-2 with the insulin-like growth factor-I receptor.

SOCS (suppressor of cytokine signaling) proteins have been shown to be negative regulators of cytokine receptor signaling via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. We have cloned a member of this family (hSOCS-2) by utilizing the insulin-like growth factor I receptor (IGF-IR) cytoplasmic domain as bait in a yeast two-hybrid screen of a human fetal brain library. The hSOCS-2 protein interacted strongly with the activated IGF-IR and not with a kinase negative mutant receptor in the two-hybrid assay. Mutation of receptor tyrosines 950, 1250, 1251, and 1316 to phenylalanine or deletion of the COOH-terminal 93 amino acids did not result in decreased interaction of the receptor with hSOCS-2 protein. hSOCS-1 protein also interacted strongly with IGF-IR in the two-hybrid assay. Glutathione S-transferase-hSOCS-2 associated with activated IGF-IR in lysates of mouse fibroblasts overexpressing IGF-IR. Human embryonic kidney cells (293) were transiently transfected with vectors containing IGF-IR and FLAG epitope-tagged hSOCS-2. After IGF-I stimulation, activated IGF-IR was found in anti-FLAG immunoprecipitates and, conversely, FLAG-hSOCS-2 was found in anti IGF-IR immunoprecipitates. Thus, hSOCS-2 interacted with IGF-IR both in vitro and in vivo. HSOCS-2 mRNA was expressed in many human fetal and adult tissues with particularly high abundance in fetal kidney and adult heart, skeletal muscle, pancreas, and liver. These results raise the possibility that SOCS proteins may also play a regulatory role in IGF-I receptor signaling.

Interleukin-12 inhibits graft-versus-host disease through an Fas-mediated mechanism associated with alterations in donor T-cell activation and expansion.

We have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12-treated mice showed marked reductions in splenic donor CD4(+) and CD8(+) T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4(+) cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12-treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12-protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12-treated group. Expression of Fas was increased on donor CD4 cells of IL-12-treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4(+) T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.

Cloning of human p55 gamma, a regulatory subunit of phosphatidylinositol 3-kinase, by a yeast two-hybrid library screen with the insulin-like growth factor-I receptor.

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular domain of the insulin-like growth factor-I receptor (IGFIR). In a search of a human fetal brain library we identified a cDNA encoding a protein that is the human homologue of mouse p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (hp55 gamma). The hp55 gamma protein interacts strongly with the activated IGFIR but not with the kinase-negative mutant receptor. hp55 gamma also interacts with the insulin receptor (IR) in the yeast two-hybrid system. The putative hp55 gamma protein is composed of a unique amino terminal region followed by a proline-rich motif and two Src homology 2 (SH2) domains, which are highly homologous to those in mouse p55PIK, rat p55 gamma, human p85 alpha and bovine p85 beta; it contains no SH3 domain. hp55 gamma mRNAs are expressed in most human fetal and adult tissues with particularly high abundance in adult testis. Splice variant(s) of hp55 gamma, one of which has a deletion of 36 amino acids at the amino terminus and another which has an insertion of 59 amino acids at position 256 between the SH2 domains, were also identified. A GST-hp55 gamma fusion protein interacts in vitro with both the activated IGFIR and IR derived from mammalian cells. Our findings suggest that hp55 gamma interacts with the IGFIR and IR and may be involved in PI 3-kinase activation by these receptors.

14-3-3 proteins interact with the insulin-like growth factor receptor but not the insulin receptor.

We have used a yeast two-hybrid system to identify proteins which bind to the cytosolic portion of the type 1 insulin-like growth factor (IGF) receptor (IGFIR) but not the insulin receptor (IR). This analysis identified 14-3-3beta and zeta proteins. 14-3-3beta also binds to the IGFIR but not the IR in vitro and 14-3-3-IGFIR complexes are present in insect cells overexpressing the IGFIR cytoplasmic domain. 14-3-3 proteins are substrates of the IGFIR in the yeast system and in vitro. The interaction of 14-3-3 with the IGFIR requires receptor-kinase activity and maps to the C-terminus of the receptor, but does not depend on tyrosine residues in this or the juxtamembrane regions. Instead, the binding maps to serine residue 1283 and requires phosphorylation of this residue. 14-3-3 proteins are phosphoserine-binding proteins which have been shown to interact directly with components of the mitogenic and apoptotic signalling pathways, suggesting that they participate in growth regulation. Our findings suggest that 14-3-3 proteins may play a role in IGFIR signal transduction and may contribute to the differences in IGF and IR signalling capabilities.

Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10.

We have used the yeast two-hybrid system to study the interaction between the IGF-I receptor and two putative substrates, IRS-1 and Shc. In addition, we have identified Grb10 as a protein that binds to the insulin-like growth factor I (IGF-I) receptor. This two-hybrid system (the interaction trap) utilizes a hybrid protein containing the LexA DNA-binding domain fused to the intracellular portion of the IGF-I receptor (LexA-IGFIR beta) and hybrids containing an activation domain fused to either IRS-1 (Ad-IRS-1), Shc (Ad-Shc), or a cDNA library. A positive interaction of LexA-IGFIR beta with the activation domain hybrid results in activation of reporter genes, LacZ and LEU2, in the yeast. Western blotting of extracts from transformed yeast demonstrated that the LexA-IGFIR beta fusion protein was expressed and phosphorylated on tyrosine residues. Coexpression of LexA-IGFIR beta with Ad-IRS-1 resulted in strong activation of both reporter genes; activation did not occur with a kinase-negative receptor mutant. IRS-1 residues 160-516 were sufficient for this strong interaction. Coexpression of LexA-IGFIR beta with Ad-Shc also resulted in strong activation of both LacZ and LEU2 reporter genes. This interaction was also dependent upon a tyrosine kinase-active receptor and required tyrosine 950 in the juxtamembrane region of the receptor. An N-terminal fragment of Shc (amino acids 1-232) interacted almost as strongly as full-length Shc whereas the Shc SH2 domain only activated the more sensitive LEU2 reporter. Full-length Shc was phosphorylated on tyrosine when coexpressed with IGFIR beta but not when coexpressed with the kinase-negative receptor mutant. To identify additional proteins that interact with the IGFIRs, a human fetal brain cDNA library was screened using the interaction trap system. This analysis identified partial cDNAs for Grb10. Coexpression of LexA-IGFIR beta with Ad-Grb10 resulted in strong activation of both LacZ and LEU2 reporter genes; this interaction was dependent upon a tyrosine kinase-active receptor but did not require tyrosine 950.