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PubMed2004

Organometallic complexes with biological molecules. XVIII. Alkyltin(IV) cephalexinate complexes: synthesis, solid state and solution phase investigations.

Dialkyltin(IV) and trialkyltin(IV) complexes of the deacetoxycephalo-sporin-antibiotic cephalexin [7-(d-2-amino-2-phenylacetamido)-3-methyl-3-cephem-4-carboxylic acid] (Hceph) have been synthesized and investigated both in solid and solution phase. Analytical and thermogravimetric data supported the general formula Alk(2)SnOHceph(.)H(2)O and Alk(3)Snceph(.)H(2)O (Alk=Me, n-Bu), while structural information has been gained by FT-IR, (119)Sn Mössbauer and (1)H, (13)C, (119)Sn NMR data. In particular, IR results suggested polymeric structures both for Alk(2)SnOHceph(.)H(2)O and Alk(3)Snceph(.)H(2)O. Moreover, cephalexin appears to behave as monoanionic tridentate ligand coordinating the tin(IV) atom through ester-type carboxylate, as well as through beta-lactam carbonyl oxygen atoms and the amino nitrogen donor atoms in Alk(2)SnOHceph(.)H(2)O complexes. On the basis of (119)Sn Mössbauer spectroscopy it could be inferred that tin(IV) was hexacoordinated in such complexes in the solid state, showing skew trapezoidal configuration. As far as Alk(3)Sn(IV)ceph(.)H(2)O derivatives are concerned, cephalexin coordinated the Alk(3)Sn moiety through the carboxylate acting as a bridging bidentate monoanionic group. Again, (119)Sn Mössbauer spectroscopy led us to propose a trigonal configuration around the tin(IV) atom, with R(3)Sn equatorial disposition and bridging carboxylate oxygen atoms in the axial positions. The nature of the complexes in solution state was investigated by using (1)H, (13)C and (119)Sn NMR spectroscopy. Finally, the cytotoxic activity of organotin(IV) cephalexinate derivatives has been tested using two different chromosome-staining techniques Giemsa and CMA(3), towards spermatocyte chromosomes of the mussel Brachidontes pharaonis (Mollusca: Bivalvia). Colchicinized-like mitoses (c-mitoses) on slides obtained from animals exposed to organotin(IV) cephalexinate compounds, demonstrated the high mitotic spindle-inhibiting potentiality of these chemicals. Moreover, structural damages such as "chromosome achromatic lesions", "chromosome breakages" and "chromosome fragments" have been identified through a comparative analysis of spermatocyte chromosomes from untreated specimens (negative controls) and specimens treated with the organotin(IV) complexes.

Animals↗
PubMed2006

Carbenoid chain reactions: substitutions by organolithium compounds at unactivated 1-chloro-1-alkenes.

The deceptively simple "cross-coupling" reactions Alk(2)C=CA-Cl + RLi --> Alk(2)C=CA-R + LiCl (A = H, D, or Cl) occur via an alkylidenecarbenoid chain mechanism in three steps without a transition metal catalyst. In the initiating step 1, the sterically shielded 2-(chloromethylidene)-1,1,3,3-tetramethylindans 2a-c (Alk(2)C=CA-Cl) generate a Cl,Li-alkylidenecarbenoid (Alk(2)C=CLi-Cl, 6) through the transfer of atom A to RLi (methyllithium, n-butyllithium, or aryllithium). The chain cycle consists of the following two steps: (i) A fast vinylic substitution reaction of these RLi at carbenoid 6 (step 2) with formation of the chain carrier Alk(2)C=CLi-R (8), and (ii) a rate-limiting transfer of atom A (step 3) from reagent 2 to the chain carrier 8 with formation of the product Alk(2)C=CA-R (4) and with regeneration of carbenoid 6. This chain propagation step 3 was sufficiently slow to allow steady-state concentrations of Alk(2)C=CLi-Aryl to be observed (by NMR) with RLi = C6H5Li (in Et2O) and with 4-(Me3Si)C6H4Li (in t-BuOMe), whereas these chain processes were much faster in THF solution. PhC[triple bond]CLi cannot perform step 1, but its carbenoid chain processes with reagents 2a and 2c may be started with MeLi, whereafter LiC[triple bond]CPh reacts faster than MeLi in the product-determining step 2 to generate the chain carrier Alk(2)C=CLi-C[triple bond]CPh (8g), which completes its chain cycle through the slower step 3. The sterically congested products were formed with surprising ease even with RLi as bulky as 2,6-dimethylphenyllithium and 2,4,6-tri-tert-butylphenyllithium.

Journal Article↗
PubMed2004

Suppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma.

Using a cDNA microarray, we found that suppressor of cytokine signaling 3 (SOCS3) is highly expressed in anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) cell lines. As SOCS3 is induced by activated signal transducer and activator of transcription 3 (STAT3), and ALK activates STAT3, we hypothesized that SOCS3 may play a role in ALK+ ALCL pathogenesis via the Janus kinase 3 (JAK3)-STAT3 pathway. Using ALCL cell lines, we show by coimmunoprecipitation experiments that SOCS3 physically binds with JAK3 in vitro, and that JAK3 inhibition by WHI-P154 downregulates SOCS3 expression. Western blot analysis confirmed expression of SOCS3 and also showed coexpression of phosphorylated (activated) STAT3 (pSTAT3). Direct sequencing of the SOCS3 gene showed no mutations or alternative splicing. In ALCL tumors that were assessed by immunohistochemistry, nine of 12 (75%) ALK+ tumors were SOCS3 positive and eight (67%) coexpressed pSTAT3. In comparison, 18 of 25 (72%) ALK-- tumors were SOCS3 positive and seven (28%) coexpressed pSTAT3. These results show that SOCS3 is overexpressed in ALCL, attributable to JAK3-STAT3 activation and likely related to ALK in ALK+ tumors. However, SOCS3 is also expressed in tumors that lack STAT3 and ALK suggesting alternative mechanisms of upregulation.

Alternative Splicing↗
PubMed2004

Bone morphogenetic protein-5, -6 and -7 inhibit growth and induce apoptosis in human myeloma cells.

Previously, bone morphogenetic protein (BMP)-2 and -4 have been shown to inhibit proliferation and induce apoptosis in human myeloma cells. BMP-2 and -4 belong to a subgroup of BMPs using the BMP receptors Alk-3 or -6. In this study, we examined the effects on human myeloma cells of BMP-6 and -7, members of a different BMP subgroup, which mainly utilize Alk-2 as their receptor. All cell lines examined expressed mRNA for the BMP-6 and -7 receptor Alk-2. We did not detect transcripts for the BMP-2 and -4 receptors Alk-3 or Alk-6 in INA-6 and RPMI-8226 cells by RT-PCR. Accordingly, the intracellular signalling molecules Smad-1, -5 and -8 were not phosphorylated by BMP-4 in INA-6 and RPMI-8226 cells. The expression patterns of various BMP receptors in the myeloma cell lines explained the differences in responses to the various BMPs. Alk-2-expressing cell lines responded with growth inhibition and apoptosis to BMP-6 and -7, whereas cell lines lacking both Alk-3 and -6 were resistant to BMP-4. Soluble Alk-3 and -6 were able to neutralize the BMP-4 effects in BMP-4-responsive cell lines. All BMPs reduced viability in more than 70% of purified primary myeloma cell samples. BMPs have intriguing antitumor effects in vitro. Importantly, myeloma cells not responsive to BMP-2 and -4 may still be sensitive to BMP-6 or -7. It is possible that therapeutic use of BMP or BMP analogues could have an impact on both myeloma bone disease and myeloma cell growth.

Apoptosis↗
PubMed2002

Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth.

Glioblastoma multiforme is the most common highly aggressive human brain cancer, and receptor tyrosine kinases have been implicated in the progression of this malignancy. We have recently identified anaplastic lymphoma kinase (ALK) as a tyrosine kinase receptor for pleiotrophin, a secreted growth factor that is highly expressed during embryonic brain development and in tumors of the central nervous system. Here we report on the contribution of pleiotrophin-ALK signaling to glioblastoma growth. We found ALK overexpressed in human glioblastoma relative to normal brain and detected ALK mRNA in glioblastoma cell lines. We reduced the endogenous ALK in glioblastoma cells by ribozyme targeting and demonstrated that this prevents pleiotrophin-stimulated phosphorylation of the anti-apoptotic protein Akt. Furthermore, this depletion of ALK reduced tumor growth of xenografts in athymic nude mice and prolonged survival of the animals because of increased apoptosis in the tumors. These findings directly implicate ALK signaling as a rate-limiting factor in the growth of glioblastoma multiforme and suggest potential utility of therapeutic targeting of ALK.

Anaplastic Lymphoma Kinase↗
PubMed2005

Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.

Anaplastic Lymphoma Kinase↗
PubMed2004

A case of primary cutaneous anaplastic large cell lymphoma with variant anaplastic lymphoma kinase translocation.

Anaplastic lymphoma kinase (ALK) is frequently observed in systemic anaplastic large cell lymphoma (ALCL), mostly in childhood or adolescence, but only rarely in primary cutaneous cases. We report a case of primary cutaneous ALCL (pcALCL) with cytoplasmic ALK expression. A 54-year-old woman with an ulcerative tumour on her forehead was admitted to our hospital. Histologically, there was an infiltrate consisting of atypical large lymphocytes and small lymphocytes in the dermis and fat tissue. Southern blot analysis showed monoclonal T-cell receptor Cbeta1 gene rearrangement. Atypical large lymphocytes were positive for CD30, CD4 and CD25, and negative for CD3 and CD79a. They were also positive for ALK only in the cytoplasm, and neurophosmin (NPM)-ALK fusion transcript was not detected by reverse transcription-polymerase chain reaction. This suggested that the translocation partner of the ALK gene in this case was different from NPM (variant translocation). The tumour on the forehead resolved in 1 month after biopsy. Nodular lesions recurred on the right knee, and were histologically identical with the forehead lesion. Our case suggests the existence of a subgroup with variant ALK translocation in pcALCL; examining NPM-ALK translocation in each case with ALK expression should be useful to characterize the disease further.

Anaplastic Lymphoma Kinase↗
PubMed2006

Absence of human herpesvirus-8 and Epstein-Barr virus in inflammatory myofibroblastic tumor with anaplastic large cell lymphoma kinase fusion gene.

Inflammatory myofibroblastic tumor (IMT) is clinically and histologically characterized by inflammation. Some populations of IMT have anaplastic large cell lymphoma kinase (ALK) gene rearrangements. Infection with Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8) in tumor cells of IMT has been reported; these reports, however, have been limited to ALK-negative IMT. The purpose of the present paper was to evaluate 21 cases of IMT for the presence of EBV and HHV-8. Immunohistochemically, 15 cases were ALK positive and six were negative. Of eight cases analyzed using reverse transcription-polymerase chain reaction, tropomyosin 3 (TPM3)-ALK, TPM4-ALK and clathrin heavy chain-ALK fusion genes were detected in one, two and two cases, respectively. All 21 IMT, irrespective of ALK expression, were negative for EBV by in situ hybridization for EBV-encoded RNA and immunohistochemical stain for latent membrane antigen-1. HHV-8 was also negative in all IMT by PCR for HHV-8 DNA sequence (KS330/233) and immunohistochemical stain for latent nuclear antigen. These results suggest that IMT may be a heterogeneous group in terms of pathogenesis, and EBV and HHV-8 do not play a major role in the pathogenesis of ALK-positive tumor.

Adolescent↗
PubMed2005

Alkalosis increases muscle K+ release, but lowers plasma [K+] and delays fatigue during dynamic forearm exercise.

Alkalosis enhances human exercise performance, and reduces K+ loss in contracting rat muscle. We investigated alkalosis effects on K+ regulation, ionic regulation and fatigue during intense exercise in nine untrained volunteers. Concentric finger flexions were conducted at 75% peak work rate (3 W) until fatigue, under alkalosis (Alk, NaHCO3, 0.3 g kg(-1)) and control (Con, CaCO3) conditions, 1 month apart in a randomised, double-blind, crossover design. Deep antecubital venous (v) and radial arterial (a) blood was drawn at rest, during exercise and recovery, to determine arterio-venous differences for electrolytes, fluid shifts, acid-base and gas exchange. Finger flexion exercise barely perturbed arterial plasma ions and acid-base status, but induced marked arterio-venous changes. Alk elevated [HCO3-] and PCO2, and lowered [H+] (P < 0.05). Time to fatigue increased substantially during Alk (25 +/- 8%, P < 0.05), whilst both [K+]a and [K+]v were reduced (P < 0.01) and [K+]a-v during exercise tended to be greater (P= 0.056, n= 8). Muscle K+ efflux at fatigue was greater in Alk (21.2+/- 7.6 micromol min(-1), 32 +/- 7%, P < 0.05, n= 6), but peak K+ uptake rate was elevated during recovery (15 +/- 7%, P < 0.05) suggesting increased muscle Na+,K+-ATPase activity. Alk induced greater [Na+]a, [Cl-]v, muscle Cl- influx and muscle lactate concentration ([Lac-]) efflux during exercise and recovery (P < 0.05). The lower circulating [K+] and greater muscle K+ uptake, Na+ delivery and Cl- uptake with Alk, are all consistent with preservation of membrane excitability during exercise. This suggests that lesser exercise-induced membrane depolarization may be an important mechanism underlying enhanced exercise performance with Alk. Thus Alk was associated with improved regulation of K+, Na+, Cl- and Lac-.

Alkalosis↗
PubMed1994

Characterization of type I receptors for transforming growth factor-beta and activin.

Transforming growth factor-beta (TGF-beta) and activin exert their effects by binding to heteromeric complexes of type I and type II receptors. The type II receptors for TGF-beta and activin are transmembrane serine-threonine kinases; a series of related receptors, denoted activin receptor-like kinase (ALK) 1 to 5, have recently been identified, and ALK-6 is described here. ALK-5 has been shown to be a functional TGF-beta type I receptor. A systematic analysis revealed that most ALKs formed heteromeric complexes with the type II receptors for TGF-beta and activin after overexpression in COS cells; however, among the six ALKs, only ALK-5 was a functional TGF-beta type I receptor for activation of plasminogen activator inhibitor-1, and only ALK-2 and ALK-4 bound activin with high affinity.

Activin Receptors↗
PubMed2004

Pancreatic trypsin cleaves intestinal alkaline sphingomyelinase from mucosa and enhances the sphingomyelinase activity.

Sphingomyelin (SM) hydrolysis in the gut has implications in colonic tumorigenesis and cholesterol absorption. It is triggered by intestinal alkaline sphingomyelinase (Alk-SMase) that is present in the intestinal mucosa and content. The mechanism by which the enzyme is released into the lumen is not clear. We studied whether trypsin can dissociate Alk-SMase from the mucosa and affect its activity. During luminal perfusion of rat intestine, addition of trypsin to the buffer increased Alk-SMase activity in the perfusate output by about threefold. Treating COS-7 cells transfected with Alk-SMase cDNA with trypsin increased the SMase activity in the medium and reduced that in the cell lysate dose dependently. The appearance of Alk-SMase in the perfusate and culture medium was confirmed by Western blot analysis. The effect of trypsin was blocked by trypsin inhibitor, and neither chymotrypsin nor elastase had a similar effect. We also expressed the full length and COOH-terminal truncated Alk-SMase in COS-7 cells and found that the activity of the full-length enzyme is mainly in the cells, whereas that of the truncated form is mainly in the medium. Both forms were active, but only the activity of the full-length Alk-SMase was enhanced by trypsin. By linking a poly-His tag to the constructed cDNA, we found that the first tryptic site Arg440 upstream of the signal anchor was attacked by trypsin. In conclusion, trypsin cleaves the Alk-SMase at the COOH terminal, releases it from mucosa, and meanwhile enhances its activity. The findings indicate a physiological role of trypsin in SM digestion.

Alkalies↗
PubMed2006

Activation of mammalian target of rapamycin signaling pathway contributes to tumor cell survival in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E-binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G(1)-S-phase regulators, including the cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL.

Anaplastic Lymphoma Kinase↗
PubMed1995

Randomized comparison of vinorelbine and melphalan in anthracycline-refractory advanced breast cancer.

PURPOSE: This prospective multicenter randomized trial was performed to compare the effectiveness and safety of intravenous (i.v.) vinorelbine tartrate (Navelbine [NVB]; Burroughs Wellcome Co, Research Triangle Park, NC) with i.v. melphalan (Alkeran [ALK]; Burroughs Wellcome Co) in a heavily pretreated population of patients with anthracycline-refractory advanced breast cancer (ABC). Efficacy end points included time to disease progression (TDP), time to treatment failure (TTF), survival, tumor response rates, and quality of life (QL) and relief of cancer-related symptoms. PATIENTS AND METHODS: Between August 24, 1990, and December 1, 1992, 183 patients were randomized (2:1) to treatment with NVB (30 mg/m2 weekly) or ALK (25 mg/m2 every 4 weeks) i.v. Patients were stratified by measurable or nonmeasurable-assessable disease and by treatment center. RESULTS: Time to disease progression was significantly longer with NVB than with ALK, with a median 12 weeks versus 8 weeks, respectively (P < .001). NVB patients also had significantly longer time to treatment failure than ALK patients, with a median 12 weeks versus 8 weeks, respectively (P < .001). The effect of NVB on survival was also statistically significant (P = .034): 1-year survival rates were 35.7% with NVB and 21.7% with ALK and the median survival rate was 35 weeks and 31 weeks, respectively. In total, 46.5% of NVB patients and 28.2% of ALK patients achieved an objective response or stabilization of disease (P = .06). No intergroup differences were noted in patient-assessed QL and cancer-related symptoms. The most common toxicities were hematologic, including granulocytopenia with NVB and thrombocytopenia and granulocytopenia with ALK. Both drugs were generally well tolerated, and no septic deaths were reported. CONCLUSION: This randomized trial demonstrates a survival benefit in anthracycline-refractory ABC. NVB was well tolerated and demonstrated activity superior to ALK in anthracycline-refractory ABC, without compromising QL. Based on activity of single-agent NVB in this difficult-to-treat patient population, investigations of NVB in combination with other anticancer drugs are warranted.

Adult↗
PubMed2004

Characterization of the bone morphogenetic protein (BMP) system in human pulmonary arterial smooth muscle cells isolated from a sporadic case of primary pulmonary hypertension: roles of BMP type IB receptor (activin receptor-like kinase-6) in the mitotic action.

The functional involvement of bone morphogenetic protein (BMP) system in primary pulmonary hypertension (PPH) remains unclear. Here we demonstrate a crucial role of the BMP type IB receptor, activin receptor-like kinase (ALK)-6 for pulmonary arterial smooth muscle cell (pphPASMC) mitosis isolated from a sporadic PPH patient bearing no mutations in BMPR2 gene. A striking increase in the levels of ALK-6 mRNA was revealed in pphPASMC compared with control PASMCs, in which ALK-6 transcripts were hardly detectable. BMP-2 and -7 stimulated the mitosis of pphPASMCs, which was opposite to their suppressive effects on the mitosis of the control PASMCs. BMP-4 and -6 and activin inhibited pphPASMC mitosis, whereas these did not affect control PASMCs. The presence of BMP signaling machinery in pphPASMCs was elucidated based on the analysis on Id-1 transcription and Smad-reporter genes. Overexpression of a dominant-negative ALK-6 construct revealed that ALK-6 plays a key role in the mitosis as well as intracellular BMP signaling of pphPASMCs. Gene silencing of ALK-6 using small interfering RNA also reduced DNA synthesis as well as Id-1 transcription in pphPASMCs regardless of BMP-2 stimulation. Although Id-1 response was not stimulated by BMP-2 in control PASMCs, the gene delivery of wild-type ALK-6 caused significant increase in the Id-1 transcripts in response to BMP-2. Additionally, inhibitors of ERK and p38 MAPK pathways suppressed pphPASMC mitosis induced by BMP-2, implying that the mitotic action is in part MAPK dependent. Thus, the BMP system is strongly involved in pphPASMC mitosis through ALK-6, which possibly leads to activation of Smad and MAPK, resulting in the progression of vascular remodeling of pulmonary arteries in PPH.

Adolescent↗
PubMed2005

Identification of anaplastic lymphoma kinase variant translocations using 5'RACE.

Anaplastic lymphoma kinase (ALK) is abnormally expressed in anaplastic large cell lymphoma (ALCL) and its expression associated with chromosomal translocations involving the ALK gene at 2p23. These translocations lead to the synthesis of novel chimeric proteins that retain the C-terminal portion of ALK, where the tyrosine kinase domain is located. In most of these tumors, the t(2;5)(p23;q35) translocation causes fusion of the ALK gene to the 5' region of the nucleophosmin (NPM) gene, but other different ALK partners have been identified, including nonmuscle tropomyosin (TPM3), TRK-fused gene (TFG), 5' aminoimidazole-4-carboxamide ribonucleotide formyltranferase/IMP cyclohydrolase (ATIC), clathrin heavy chain gene (CLTC), and moesin (MSN). The characterization of these ALK partners has been performed using different molecular methods, including the 5' Rapid Amplification of complementary deoxyribonucleic acid (cDNA) Ends (5'RACE) polymerase chain reaction (PCR)-based technique. This approach allows the potential amplification and identification of either 5' or 3' mRNA ends from an internal known sequence. In ALK translocations, identification of the 5' gene involved has been performed using primers designed within the known 3' catalytic domain of the ALK. Initial reaction consists in a first-strand cDNA synthesis primed using a gene-specific antisense primer (ALK1), performing the cDNA conversion of specific messenger ribonucleic acids, and maximizing the potential for complete extension to the 5'-end of the message. After cDNA synthesis, the first-strand product is purified from unincorporated dNTPs and ALK1. Terminal deoxynucleotidyl transferase is used to add homopolymeric tails to the 3' ends of the cDNA. Tailed cDNA is then amplified by PCR using a nested gene-specific primer (ALK2), which anneals 3' to ALK1, and a complementary homopolymer containing an anchor primer (i.e., AAP), which permits amplification from the homopolymeric tail. This allows amplification of unknown sequences between the ALK2 and the 5' end of the mRNA. Further, nested PCRs usually are required to confer an adequate level of specificity to the process to permit the characterization of RACE products. The reamplification is achieved by using a nested gene-specific primer (ALK3), which anneals 3' to ALK2, and a universal amplification primer, which anneals to the 5' sequence previously introduced by the AAP primer.

Anaplastic Lymphoma Kinase↗
PubMed2005

Anaplastic lymphoma kinase and its signalling molecules as novel targets in lymphoma therapy.

A crucial issue in the development of molecularly-targeted anticancer therapies is the identification of appropriate molecules whose targeting would result in tumour regression with a minimal level of systemic toxicity. Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase, normally expressed at low levels in the nervous system. As a consequence of chromosomal translocations involving the alk gene (2p23), ALK is also aberrantly expressed and constitutively activated in approximately 60% of CD30+ anaplastic large cell lymphomas (ALCLs). Due to the selective overexpression of ALK in tumour cells, its direct involvement in the process of malignant transformation and its frequent expression in ALCL patients, the authors recognise ALK as a suitable candidate for the development of molecularly targeted strategies for the therapeutic treatment of ALK-positive lymphomas. Strategies targeting ALK directly or indirectly via the inhibition of the protein networks responsible for ALK oncogenic signalling are discussed.

Anaplastic Lymphoma Kinase↗
PubMed2002

Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma.

Accumulating evidence indicates that expression of anaplastic lymphoma kinase (ALK), typically due to t(2;5) translocation, defines a distinct type of T/null-cell lymphoma (TCL). The resulting nucleophosmin (NPM) /ALK chimeric kinase is constitutively active and oncogenic. Downstream effector molecules triggered by NPM/ALK remain, however, largely unidentified. Here we report that NPM/ALK induces continuous activation of STAT3. STAT3 displayed tyrosine phosphorylation and DNA binding in all (four of four) ALK+ TCL cell lines tested. The activation of STAT3 was selective because none of the other known STATs was consistently tyrosine phosphorylated in these cell lines. In addition, malignant cells in tissue sections from all (10 of 10) ALK+ TCL patients expressed tyrosine-phosphorylated STAT3. Transfection of BaF3 cells with NPM/ALK resulted in tyrosine phosphorylation of STAT3. Furthermore, STAT3 was constitutively associated with NPM/ALK in the ALK+ TCL cell lines. Additional studies into the mechanisms of STAT3 activation revealed that the ALK+ TCL cells expressed a positive regulator of STAT3 activation, protein phosphatase 2A (PP2A), which was constitutively associated with STAT3. Treatment with the PP2A inhibitor calyculin A abrogated tyrosine phosphorylation of STAT3. Finally, ALK+ T cells failed to express a negative regulator of activated STAT3, protein inhibitor of activated STAT3. These data indicate that NPM/ALK activates STAT3 and that PP2A and lack of protein inhibitor of activated STAT3 may be important in maintaining STAT3 in the activated state in the ALK+ TCL cells. These results also suggest that activated STAT3, which is known to display oncogenic properties, as well as its regulatory molecules may represent attractive targets for novel therapies in ALK+ TCL.

Anaplastic Lymphoma Kinase↗
PubMed2006

[Diffuse large B-cell lymphoma with expression of anaplastic lymphoma kinase protein: clinicopathologic and immunohistochemical study of 5 cases].

OBJECTIVE: To study the clinicopathologic features of diffuse large B-cell lymphoma (DLBCL) with expression of anaplastic lymphoma kinase (ALK) protein. METHODS: Nine hundred and forty-five (945) cases of DLBCL (including 177 consultation cases) diagnosed according to the 2001 World Health Organization classification of tumors of hematopoietic and lymphoid tissues were enrolled into the study. Immunohistochemical study for anti-ALK-11 was performed using LSAB technique. The ALK-positive cases were further confirmed by immunohistochemical study using EnVision technique. Only ALK-positive cases by EnVision technique were further analyzed by immunostaining for antigens including CD20, CD3, CD30, EMA, granzyme-B, TIA-1 and PC. Immunoglobulin heavy chain gene rearrangement study was also performed and follow-up data collected. RESULTS: There were altogether 5 (4 males and 1 female) cases of DLBCL showing expression of ALK protein. The age of the patients ranged from 34 to 72 years. All were primary nodal DLBCL. One case belonged to clinical stage I, 2 in stage II and 2 in stage III. The duration of follow up ranged from 4 to 32 months. Three patients subsequently died and the longest survival was 32 months. Morphologic subtypes included centroblastic 2, anaplastic 1, immunoblastic with plasmacytoid differentiation 1 and plasmablastic 1. Immunohistochemically, 4 cases were CD20 positive (including 2 centroblastic, 1 anaplastic and 1 immunoblastic cases). The plasmablastic case expressed kappa light chain and was negative for CD20. Rearrangement of immunoglobulin heavy chain gene was demonstrated in all 5 cases studied. As for ALK protein staining, a mixed membranous and cytoplasmic (1 immunoblastic case), granular cytoplasmic (2 centroblastic and 1 anaplastic cases) and mixed nuclear and cytoplasmic (1 plasmablastic case) patterns were observed. CONCLUSIONS: Expression of ALK protein is a rare phenomenon in DLBCL and can be seen in centroblastic, anaplastic, immunoblastic and plasmablastic subtypes. It is often associated with aggressive clinical behavior and worse prognosis. A new pattern of ALK protein expression, mixed membranous and cytoplasmic, is reported.

Adult↗