Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors.

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo- and targeted therapies.

Analysis of G(-174)C IL-6 polymorphism and plasma concentrations of inflammatory markers in patients with type 2 diabetes and peripheral arterial disease.

AIMS: To determine whether the G(-174)C interleukin 6 (IL-6) polymorphism influences the development of peripheral arterial disease (PAD) in individuals with type 2 diabetes. This was investigated by comparing the distribution of G(-174)C genotypes between patients with type 2 diabetes and PAD (PAD+) and those with type 2 diabetes but without PAD (PAD-). Plasma concentrations of IL-6, fibrinogen, C reactive protein (CRP), and vascular endothelial growth factor (VEGF) were also compared in PAD+ and PAD- patients. METHODS: Blood samples were collected from 146 PAD+ and 144 PAD- patients. SfaNI was used to determine the G(-174)C genotype. Plasma concentrations of IL-6, fibrinogen, CRP, and VEGF were measured by an enzyme linked immunosorbent assay. RESULTS: The GG genotype was more common in PAD+ patients than in PAD- patients. PAD+ patients also had increased mean plasma concentrations of IL-6, fibrinogen, CRP, and VEGF compared with PAD- patients. Mean plasma concentrations of IL-6, fibrinogen, and CRP in both PAD+ and PAD- patients were higher in those with the GG genotype than in those with the GC or CC genotypes. In contrast, mean plasma concentrations of VEGF in PAD+ and PAD- patients were not significantly different between those with different G(-174)C genotypes. CONCLUSIONS: These results support a model in which the GG genotype promotes PAD development among individuals with type 2 diabetes by inducing increased release of IL-6. Higher concentrations of IL-6 among those with the GG genotype is associated with increased plasma concentrations of fibrinogen and CRP.

Analysis of hepatitis C virus infection among health-care workers: an observational study.

AIM: Hepatitis C virus (HCV) is one of the most common blood-borne pathogens transmitted from patients to health care workers (HCWs). The Centers for Disease Control and Prevention (CDC) have developed a set of universal precautions to help prevent transmission of blood-borne pathogens between patients and HCWs in health care settings. HCV infection status among HCWs and proportion of HCWs experiencing occupational blood exposure accidents were monitored to assess the risk of HCV infection among HCWs at a hospital in Catania, Italy. METHODS: The number of HCWs reporting occupational blood exposure accidents during 1999 and 2004 were compared to examine whether there was any change in the incidence of these accidents among 900 HCWs. HCV infection status of these HCWs was also analyzed in 1999 and 2004 to determine how many were infected with HCV during this time period. RESULTS: HCV infection was detected in 21 out of 900 subjects in 1999. The remaining 879 HCWs remained HCV-negative until they were last tested in 2004. There was a statistically significant decrease in the number of HCWs that experienced occupational blood exposure accidents from 306 in 1999 to 240 in 2004 (P = 0.001). CONCLUSIONS: The finding that all 871 HCV-negative HCWs remained HCV-negative from 1999 until 2004 supports the view that the set of universal precautions recommended by the CDC are helpful for preventing HCV transmission from patients to HCWs. HCWs must continue following these precautions to prevent transmission of HCV and other blood-borne pathogens between patients and HCWs in the future.

Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention.

The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression. Thus, it is an appropriate pathway to target for therapeutic intervention. This pathway becomes more complex daily, as there are multiple members of the kinase and transcription factor families, which can be activated or inactivated by protein phosphorylation. The diversity of signals transduced by this pathway is increased, as different family members heterodimerize to transmit different signals. Furthermore, additional signal transduction pathways interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways (eg PI3K, PTEN, Akt), which serve to regulate its activity. Dysregulation of this pathway can result in autocrine transformation of hematopoietic cells since cytokine genes such as interleukin-3 and granulocyte/macrophage colony-stimulating factor contain the transacting binding sites for the transcription factors regulated by this pathway. Inhibitors of Ras, Raf, MEK and some downstream targets have been developed and many are currently in clinical trials. This review will summarize our current understanding of the Ras/Raf/MEK/ERK signal transduction pathway and the downstream transcription factors. The prospects of targeting this pathway for therapeutic intervention in leukemia and other cancers will be evaluated.

Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia.

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DeltaMEK1:ER, a conditionally active form of MEK1 which responds to either beta-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DeltaMEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70(S6K)) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of beta-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DeltaMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70(S6K) activation. Treatment with PI3K, Akt and p70(S6K) inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70(S6K) inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DeltaAkt(Myr(+))], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.

Elitek-rasburicase: an effective means to prevent and treat hyperuricemia associated with tumor lysis syndrome, a Meeting Report, Dallas, Texas, January 2002.

Renal precipitation of uric acid associated with tumor lysis syndrome (TLS) is a major complication in the management of leukemia, lymphoma, and other drug-sensitive cancers. Management of hyperuricema has historically consisted of administration of allopurinol, hydration, alkalinization to maintain pH between 7.0 and 7.3, and in some cases diuresis. Allopurinol, a xanthine analogue, blocks xanthine oxidase and formation of uric acid. Urate oxidase converts uric acid to allantoin, which is 5-10 times more soluble than uric acid. Homo sapiens cannot express urate oxidase because of a nonsense mutation. Urate oxidase was initially purified from Aspergillus flavus fungus. Treatment with this nonrecombinant product had been effective in preventing renal precipitation of uric acid in cancer patients, but was associated with a relatively high frequency of allergic reactions. This enzyme was recently cloned from A. flavus and is now manufactured as a recombinant protein. Clinical trials have shown this drug to be more effective than allopurinol for prevention and treatment of hyperuricemia in leukemia and lymphoma patients. This drug has been approved in Europe as well as the US and several clinical trials are in progress to further determine its clinical utility in other patient subsets. The purpose of this meeting was to discuss usefulness of recombinant urate oxidase, also known as rasburicase, Fasturtec, and Elitek, for the management of TLS in certain cancer patients.

Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy.

The PI3K/Akt signal transduction cascade has been investigated extensively for its roles in oncogenic transformation. Initial studies implicated both PI3K and Akt in prevention of apoptosis. However, more recent evidence has also associated this pathway with regulation of cell cycle progression. Uncovering the signaling network spanning from extracellular environment to the nucleus should illuminate biochemical events contributing to malignant transformation. Here, we discuss PI3K/Akt-mediated signal transduction including its mechanisms of activation, signal transducing molecules, and effects on gene expression that contribute to tumorigenesis. Effects of PI3K/Akt signaling on important proteins controlling cellular proliferation are emphasized. These targets include cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors. Furthermore, strategies used to inhibit the PI3K/Akt pathway are presented. The potential for cancer treatment with agents inhibiting this pathway is also addressed.

Role of the Raf signal transduction cascade in the in vitro resistance to the anticancer drug doxorubicin.

The precise molecular events involved in the development of drug resistance (DR) remain largely unknown. Raf is an intermediate in the signal transduction cascades initiated by growth factors. The hypothesis behind the following studies is that deregulated Raf-1 expression plays a role in the development of drug resistance. A positive correlation was observed between increased Raf-1 activity and increased values for IC50 for doxorubicin in established cell lines. The National Cancer Institute/Adriamycin Resistant (NCI/ADR-RES) cell line exhibited both the highest Raf-1 activity and the highest IC50 values for doxorubicin (Adriamycin). In contrast, the MCF-7 cell line exhibited both lower Raf activity and lower IC50 values for doxorubicin. While MCF-7 cells transfected with either constitutively active DeltaRaf-1 or conditionally active DeltaRaf-1:AR demonstrated increased IC50 values for doxorubicin and a reduced capacity to undergo apoptosis after doxorubicin treatment as compared with parental cell lines. Moreover, growth curves performed show that both the constitutively and conditionally active forms of Raf-1 do not increase growth as compared with the parental MCF-7 cell line. This is important because it implies that higher cell counts between Raf transfectants and the parental MCF-7 cell line are attributable to differences in DR, not growth rates. These observations suggest a role for the Raf-1 protooncogene in the regulation of DR.

Identification of isoforms of a mitotic motor in mammalian spermatogenesis.

We have isolated the full-length coding sequence for mouse KIFC5A (kinesin family c-terminal 5A) cDNA, encoding a motor protein found in the testes. The complete sequence of the KIFC5A cDNA is homologous to a group of carboxyl-terminal motors, including hamster CHO2, human HSET, and mouse KIFC1 and KIFC4. The KIFC5A and KIFC1 cDNAs are nearly identical except for the presence of two additional sequence blocks in the 5'-end of KIFC5A and a number of single base-pair differences in their motor domains. Polymerase chain reaction amplification and sequencing of the 5'-end of KIFC5A identified 3 distinct RNA species in testes and other tissues. Sequence comparison and genetic mapping confirmed the existence of a small multi-gene family in the mouse and suggest possible mechanisms of alternative splicing, genetic duplication, and separate genetic loci in the generation of these motors. In order to examine the possible role of these motors in germ cells of the testes, an antibody to a shared epitope was used to localize this group of proteins to different spermatogenic cell types. These experiments suggest that KIFC5-like motor proteins are associated with multiple microtubule complexes in male germ cells, including the meiotic spindle, the manchette, and the flagella.