Establishment and characterization of an STI571-resistant human myelogenous leukemia cell line, SR-1.

Posted by rob on October 22, 2004 under Uncategorized | Be the First to Comment

Cancer Genet Cytogenet. 2004 Oct 1; 154(1): 52-6
Kwon HC, Kim SH, Kim JS, Han H, Roh MS, Han JY, Seo SY, Lee YH, Kim HJ

The tyrosine kinase inhibitor STI571 is an effective agent for the treatment of chronic myelogenous leukemia (CML). However, a lack of response to STI571 or the recurrence of the disease after a transient initial response is usually seen in patients with advanced stage CML. We have established a novel STI571 (Gleevec/Glivec, imatinib mesylate)-resistant acute myelocytic leukemia cell line (SR-1) from an STI571-resistant blast crisis patient. By flow cytometry, the immunophenotype of SR-1 was found to be compatible with a myeloid lineage (CD13+, CD33+, HLA-DR+, anti-MPO+). Conventional cytogenetics showed a three-way reciprocal translocation involving 7p22, 9q34, and 22q11.2, i.e., a variant Philadelphia chromosome translocation. The BCR/ABL rearrangement was detected by fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction. To determine the tumorigenicity of the SR-1 cell line in vivo, cells were injected subcutaneously into severe combined immunodeficiency mice. Four weeks later, tumors had grown and showed the same laboratory findings as in SR-1. Although STI571 resistance is a known treatment complication, in vivo STI571-resistant cell lines have not been fully established. We hope that our SR-1 cell line may be useful in molecular pathogenetic investigations of STI571-resistant CML.

http://www.hubmed.org/display.cgi?issn=01654608&uids=15381372

Imatinib (STI571)-mediated changes in glucose metabolism in human leukemia BCR-ABL-positive cells.

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Clin Cancer Res. 2004 Oct 1; 10(19): 6661-8
Gottschalk S, Anderson N, Hainz C, Eckhardt SG, Serkova NJ

The therapeutic efficacy of imatinib mesylate (Gleevec) is based on its specific inhibition of the BCR-ABL oncogene protein, a widely expressed tyrosine kinase in chronic myelogenous leukemia (CML) cells. The goal of this study was to evaluate glucose metabolism in BCR-ABL-positive cells that are sensitive to imatinib exposure. Two human BCR-ABL-positive cell lines (CML-T1 and K562) and one BCR-ABL-negative cell line (HC-1) were incubated with different imatinib concentrations for 96 hours. Magnetic resonance spectroscopy on cell acid extracts was performed to evaluate [1-13C]glucose metabolism, energy state, and changes in endogenous metabolites after incubation with imatinib. Imatinib induced a concentration-dependent inhibition of cell proliferation in CML-T1 (IC50, 0.69 +/- 0.06 micromol/L) and K562 cells (IC50, 0.47 +/- 0.04 micromol/L), but not in HC-1 cells. There were no metabolic changes in imatinib-treated HC-1 cells. In BCR-ABL-positive cells, the relevant therapeutic concentrations of imatinib (0.1-1.0 micromol/L) decreased glucose uptake from the media by suppressing glycolytic cell activity (C3-lactate at 0.25 mmol/L, 65% for K562 and 77% for CML-T1 versus control). Additionally, the activity of the mitochondrial Krebs cycle was increased (C4-glutamate at 0.25 micromol/L, 147% for K562 and 170% for CML-T1). The improvement in mitochondrial glucose metabolism resulted in an increased energy state (nucleoside triphosphate/nucleoside diphosphate at 0.25 micromol/L, 130% for K562 and 125% for CML-T1). Apoptosis was observed at higher concentrations. Unlike standard chemotherapeutics, imatinib, without cytocidal activity, reverses the Warburg effect in BCR-ABL-positive cells by switching from glycolysis to mitochondrial glucose metabolism, resulting in decreased glucose uptake and higher energy state.

http://www.hubmed.org/display.cgi?issn=10780432&uids=15475456

Histone deacetylase inhibitor NVP-LAQ824 has significant activity against myeloid leukemia cells in vitro and in vivo.

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Leukemia. 2004 10 21;
Weisberg E, Catley L, Kujawa J, Atadja P, Remiszewski S, Fuerst P, Cavazza C, Anderson K, Griffin JD

NVP-LAQ824 is a novel potent hydroxamic acid-derived histone deacetylase inhibitor that induces apoptosis in nanomolar concentrations in myeloid leukemia cell lines and patient samples. Here we show the activity of NVP-LAQ824 in acute myeloid leukemia cells and BCR/ABL-expressing cells of mouse and human origin, both sensitive and resistant to imatinib mesylate (Gleevec, STI-571). Whereas imatinib inhibited overall cellular tyrosine phosphorylation in Ba/F3.p210 cells, NVP-LAQ824 did not inhibit tyrosine phosphorylation, and did not affect BCR/ABL or ABL protein expression. Neither compound was able to inhibit cellular tyrosine phosphorylation in the imatinib-resistant Ba/F3.p210-T315I cell line. These data taken together suggest that BCR/ABL kinase activity is not a direct target of NVP-LAQ824. Synergy between NVP-LAQ824 and imatinib was demonstrated against BCR/ABL-expressing K562 myeloid leukemia cell lines. In addition, we show that NVP-LAQ824 was well tolerated in vivo in a pre-clinical murine leukemia model, with antileukemia activity resulting in significant prolongation of the survival of mice when treated with NVP-LAQ824 compared to control mice. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in myeloid malignancies.Leukemia advance online publication, 21 October 2004; doi:10.1038/sj.leu.2403519.

http://www.hubmed.org/display.cgi?issn=08876924&uids=15496979

Tyrosine kinase inhibitor AG1024 exerts antileukaemic effects on STI571-resistant Bcr-Abl expressing cells and decreases AKT phosphorylation.

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Br J Cancer. 2004 10 19;
Deutsch E, Maggiorella L, Wen B, Bonnet ML, Khanfir K, Frascogna V, Turhan AG, Bourhis J

Chronic myelogenous leukaemia (CML) is a clonal malignancy of the pluripotent haematopoietic stem cell, characterised by an uncontrolled proliferation and expansion of myeloid progenitors expressing a fusion oncogene, BCR-ABL, the molecular counterpart of the Ph1 chromosome. The tyrosine kinase (TK) activity of BCR-ABL is known to activate several major signalling pathways in malignant cells, including Ras, JAK/STAT and PI3K/Akt with evidence of proteasome-mediated degradation of other targets such as the DNA repair protein DNA-PKcs and cyclin-dependent kinases inhibitor p27. Targeting these abnormalities by blocking TK of BCR-ABL with STI571 provided a promising approach for the therapy of CML. The recent development of resistance to STI571 illustrates, however, that the use of other TK inhibitors could be of major interest for therapeutic purposes. To this end, the TK inhibitor Tyrphostin AG1024 was used to evaluate effect on regulation of BCR-ABL expression, inhibition of cell proliferation and tumour formation in vivo in human and murine BCR-ABL expressing cell lines. Tyrphostin AG1024 was shown to downregulate expression of BCR-ABL and P-Akt, and to upregulate DNA-PKcs expression. In addition, Tyrphostin AG1024 was able to inhibit cell proliferation, and delay tumour growth in vivo. Thus, AG1024 is able to interfere with three major targets of BCR-ABL in leukaemic cells. Interestingly, Tyrphostin AG1024 was also effective against cells resistant to STI571 by distinct mechanisms including Bcr-Abl mutation. Therefore, these data suggest that Tyrphostin AG1024 could represent the basis of a novel therapy for STI571 refractory CML.British Journal of Cancer advance online publication, 19 October 2004; doi:10.1038/sj.bjc.6602190 www.bjcancer.com.

http://www.hubmed.org/display.cgi?issn=00070920&uids=15494718

Bcr (breakpoint cluster region) protein binds to PDZ-domains of scaffold protein PDZK1 and vesicle coat protein Mint3

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J Cell Sci. 2004 10 19;
Malmberg EK, Andersson CX, Gentzsch M, Chen JH, Mengos A, Cui L, Hansson GC, Riordan JR

The breakpoint cluster region protein (Bcr) is a large soluble oligomeric multidomain protein best known because of its involvement in chronic myelogenous leukemia (CML). A chromosomal translocation between its gene and that of the c-abl kinase (‘Philadelphia chromosome’) plays a major causative role in that malignancy. Thus most attention has been paid to the role of the protein in hemopoietic cells. However, Bcr is also expressed in other cell types including epithelia. Bcr is generally considered to be a cytoplasmic protein but in addition to its kinase and GTPase exchange and activating domains it contains potentially membrane-interacting pleckstrin homology and C2 domains as well as a PDZ-binding C terminus mediating an interaction with a PDZ-domain protein at intercellular junctions of epithelial cells. We have examined the ability of Bcr to interact with other epithelial PDZ proteins and found specific binding to both the apical PDZK1 protein and the Golgi-localized Mint3. The former is important in the organization of several apical functions and the latter in vesicular trafficking in the secretory pathway. Hence these findings extend the interactions and likely signaling impact of Bcr in epithelia from the cytosol to at least these two membrane compartments.

http://www.hubmed.org/display.cgi?issn=00219533&uids=15494376

Letter from the Affordable Medicines Treatment Campaign to Indiaâ??s National Human Rights Commission

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11th October 2004  
 
Justice A S Anand  
The Chairperson  
National Human Rights Commission (NHRC)  
Sardar Patel Bhavan, Sansad Marg  
New Delhi 110001  
 
Sub: (Third Amendment to Patents Act 2004)

Dear Justice Anand:  
 
I am writing this letter on behalf of Affordable Medicines and Treatment Campaign (AMTC). The AMTC is a national campaign aimed at creating an environment that will ensure sustained accessibility and affordability of medicines and treatment for every individual in India, including access to affordable Anti-Retroviral Therapy for persons living with HIV/AIDS. It consists of civil society organisations, NGOs, patients groups, healthcare providers and concerned individuals. The campaign was initiated in 2001 with the following mission statement:  
 

    The right to life and health is a fundamental right guaranteed to every person living in India and is non-negotiable. This campaign aims to demand and create an environment that will ensure sustained accessibility and affordability of medicines and treatment for every individual in India, including access to affordable Anti-Retroviral Therapy for persons living with HIV/AIDS. This campaign shall be democratic and participatory. It will seek the mobilization of communities and civil society to make state, national and international agencies and industry accountable for securing health for all.

 
 
AMTC attempts to encourage action on a wide spectrum of issues relating to the right to treatment. These include WTO negotiations relating to the TRIPS agreement (TRIPS), the impending amendment to Patents Act 1970, law and regulation of drug pricing, national and state level governmental policies relating to health including vertical disease programmes, health needs of the population, transparency and accountability in the pharmaceutical sector, treatment literacy and health care infrastructure etc.  
 
As you are aware the Government of India has initiated to amend the Patents Act 1970 to introduce product patent protection to drug, medicine or food. A Patent Amendment Bill (Bill) was introduced to this effect in the 13th Lok Sabha in December 2003. The Bill lapsed due to the dissolution of Lok Sabha. In the last week of August 2004 the Cabinet has decided to refer the Bill to a Group of Ministers (GOM) to study the implications of contentious issues in the Bill. The GOM consists of Minister of Defense, Minister of Health, Minister of Human Resources Development and Minister of Commerce and Industry. We have learned from news reports that provisions of the referred Bill are identical to the lapsed Bill. This is a matter of concern because the Bill in its present form seriously compromises the accessibility and availability of medicines, two important components of right to health.  
 
Introduction of product patent protection for medicines and agro-chemicals is part of India’s obligation under (TRIPS). TRIPS obligates member countries to provide a universal minimum standard of protection for various types intellectual property rights viz. trade mark, copyrights, geographical indications, patents, industrial designs, plant varieties, topography of integrated circuits and trade secrets. India has amended the  
Patents Act in 1999 and 2002 to comply with the obligations of TRIPS. The only pending obligation with regard to TRIPS is the introduction of product patents to medicines and agro-chemicals. India is to carry out this obligation by, before 1st January 2005.  
 
As you know, the Patents Act 1970 does not provide product patent protection for medicines and food. The Patents Act 1970 was enacted to scrap the Patents Act 1911, which provided product patent protection for medicines and food. The Patents Act 1970 provides only process patent protection to medicines and food. Process patent does not prevent others from making the product per se but prevents others from using the patented process as well as using, offering for sale, selling or importing the product obtained from the patented process. On the other hand, product patent prohibits others from making, using, offering for sale, selling or importing the patented product. As a result the product patent gives a monopoly to the patent owner for the production of patented article during the term of the patent (20 years). Therefore product patent protection for medicines and agro-chemicals creates monopoly and eliminates competition in the pharmaceutical market. Drug companies often abuse the patent monopoly and fix exorbitant prices for the patented medicines. The introduction of product patent thus reduces accessibility and affordability of drugs.  
 
The impact of product patent on the accessibility and availability of drugs are well documented. The most striking incident is the impact of product patent on access to HIV/AIDS drugs. Till 2000, antiretroviral (ARV) drugs were not accessible to the vast majority of people living with HIV/AIDS (PLHA) all over the world because of the high price. Multinational drug companies priced ARV drugs between US$12-13,000 annually per person. The price started falling in 2000 when manufacturers from India introduced generic versions of ARV drugs. Now these generic drugs are given as low as US$ 140 annually per person to certain international organisations like the Clinton Foundation. This is possible because of the absence of a product patent regime in India. Further, the absence of product patent protection has also facilitated the introduction of fixed dose combination (FDC) of ARV drugs. A three-in-one cocktail pill introduced by the generic manufacturers substituted two pills for six pills per day. Thus the FDCs increased the accessibility as well as availability of ARV drugs. The introduction of FDCs became possible only because of the absence of product patent protection in India. The introduction of a product patent regime would prevent generic companies in India from repeating this miracle.  
 
The impact of monopoly on access to medicines is already felt in India. The Controller of Patents has granted an Exclusive Marketing Right (EMR) to Novartis AG, for the drug called Gleevec used for the treatment of patients suffering from Chronic Myeloid Leukaemia (CML), a life threatening form of cancer. EMR is granted as a transitional arrangement before providing product patent protection. Gleevec is sold by Novartis AG at Rs. 1,20,000 per month.. The generic version of the drug was otherwise available to CML patients at Rs 9,000- 12,000 per month. The EMR, if enforced will result in the withdrawal of generic version of Gleevec from the market. Consequently, the overwhelming majority of patients that suffer from CML every year in India will be denied access to this life saving drug. Both the industry and the civil society have approached the Supreme Court of India to challenge the decision of granting EMR on Gleevec.  
 
Thus it shows that introduction of product patent in India will reduce the accessibility and availability of medicines. Therefore, we feel that utmost care is required to introduce the product patent regime. Certain flexibility available under the TRIPS should be used to its full extent to safeguard accessibility and availability of drugs and medicines. The Bill, however, fails to make use of this flexibility at its optimum level. Main criticisms against the bill are as follows.  
 
Firstly, the Bill proposes to extend the scope of patentability beyond the TRIPS requirements by amending Section 3 (d) to allow patent protection for new use of known drugs. Patent for new use would help pharmaceutical companies to extend the monopoly over the drug even after the expiry of original patent. There is no obligation under TRIPS to provide patent protection to new use of known drugs. Earlier, the Mashelkar Committee recommended to limit the patent protection only to new chemical molecules.  
 
Secondly, the Bill proposes to do away with the pre-grant opposition procedure. Currently, there are approximately 6,000 applications pending in the mailbox. In the absence of pre-grant opposition, these 6,000 applications would escape much needed public scrutiny. Public scrutiny is crucial in light of the fact that less than 500 drugs have been granted marketing approvals in India between 1995-2004.  
 
Thirdly, the Bill has not properly incorporated the “August 30th Decision”, which permits the grant of compulsory license for export purposes. The Bill proposes to permit compulsory licensing for export purpose if there is a compulsory license in the importing country having no or insufficient manufacturing capacity in the pharmaceutical sector. This ignores the fact that Least Developing Countries (LDCs) need not provide product patent till 2016. In the absence of patent protection, issuance of compulsory license is impossible. In that event, the Indian drug companies would not be able to export to LDCs.  
 
Lastly, the Bill fails to revamp the compulsory licensing mechanism. Even though the Chapter on compulsory licenses in the Patents Act 1970 states the need for protecting the public interest, the same spirit is not reflected in the substantial provisions. Cumbersome procedures without any time line for the final disposal of application makes the compulsory license mechanism an impractical option to curb abuse of patent monopoly.  
 
India as a party to the International Covenant on Economic Social and Cultural Rights (ICESCR) has an international obligation to protect peoples’ right to health. According to Article 12 of ICESCR “The States Parties to the present Covenant recognize the right of everyone to the enjoyment of the highest attainable standard of physical and mental health”. As you know, accessibility to and availability of drugs are recognized as important components of right to health. The introduction of product patent in the current form will further reduce the access to drugs. The Supreme Court of India recognized the enforceability of right to health within the scope of Article 21 of the Indian Constitution (Vincent Panikurlangara v Union of India 1987 (2) SCC 165). Further, Article 15(1)(b) of the ICESCR recognises “right of everyone to enjoy the benefits of scientific progress and its applications”. The Supreme Court often interpreted fundamental rights in consonance with international treaties. Hence, the implementation of product patent should not result in the denial of rights guaranteed under the Constitution of India and the ICESCR. According to Section 2 (d) & (f) of Protection of Human Rights Act, 1993 rights under the ICESCR are the rights that the National Human Rights Commission (NHRC) has to protect.  
 
We therefore request your urgent intervention to safeguard peoples’ right to health guaranteed under the Constitution of India and ICESCR by using the mandate under Section 12 (d), (f) and (j) of the Protection of Human Rights Act, 1993. In the context, we request NHRC:  
 

  • To seek information from the Government of India regarding the steps which are already taken or under consideration to safeguard the right to health under the product patent regime.  
     
  • To study and recommend to the Government of India on the steps to be taken to ensure the enjoyment of benefits of scientific progress and its application, an obligation under Article 15(1) (b) of ICESCR.  
     
  • To study and recommend to the Government of India on the options available within the TRIPS Agreement to safeguard right to health.  
     
  • To hold a national level consultation with concerned individuals and groups on the implications of intellectual property rights on peoples’ right to health.

 
 
With warm regards,  
 
For AMTC  
Anand Grover  
Project Director, Lawyers collective HIV/AIDS Unit  
 
Cc: Justice Y. Bhaskar Rao , Member, NHRC  
Cc: Mr. R.S. Kalha, Member, NHRC  
Cc: Mr. P.C.Sharma, Member, NHRC  
Cc: Mr. Tarlochan Singh, Member, NHRC  

http://hrw.org/english/docs/2004/10/22/india9556.htm

Pic Of The Day

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The all-new Jeep Grand Cherokee moves up the side of a 30-story skyscraper in New York, Thursday, Oct. 21, 2004, to promote its performance, style and off-road capability. The event, staged by mounting the jeep on a track on the building, kicked-off the arrival of the 2005 Jeep Grand Cherokee at dealerships nationwide this weekend.