Addition of the Akt inhibitor triciribine overcomes antibody resistance in cells from ErbB2/Neu-positive/PTEN-deficient mammary tumors

Abstract Abstract 3152 We have reported that treatment of B-NHL cell lines with rituximab resulted in the inhibition of the constitutively activated PI3K-AKT pathway (Suzuki et al., Oncogene 26:6184, 2007). Examination of the mechanism by which rituximab inhibits the PI3K/Akt pathway revealed that it induces the expression of the PI3K/Akt inhibitor PTEN (phosphatase and tensin homolog detected on chromosome 10). Time kinetic analysis indicated that the induction of PTEN occurs as early as 6 h post-rituximab treatment. The objective of this study is to delineate the molecular mechanism by which PTEN is induced by rituximab. We hypothesized that rituximab-induced inhibition of the constitutively activated NF-κB pathway, directly and indirectly through inhibition of the PI3K/Akt pathway, may result in the inhibition downstream of the PTEN transcription factors and repressors, Snail and Yin Yang 1 (YY1). Snail has been reported to repress the transcription of PTEN (Escriva, M et al., Mol Cell Biol 28:1528, 2008). Also, YY1 has been reported to positively regulate Snail transcription and expression (Palmer, MB et al., Mol Cancer Res 7:221, 2009). In addition, the induction of PTEN by rituximab also results, in a feed-back loop, in the suppression of YY1 and Snail and potentiates the induction of PTEN (Petriella et al, Cancer Biology Therapy, 8, 1389, 2009). This hypothesis was tested using the B-NHL Ramos cells, as model, for these studies. Treatment of Ramos with rituximab (20ug/ml for 16 hours) resulted in the inhibition of NF-κB, Snail, and YY1 and induction of PTEN expression as assessed by western. The direct role of Snail and YY1 in the suppression of PTEN expression was demonstrated in cells transfected with Snail or YY1 siRNA. The treated cells demonstrated significant induction of PTEN and, concomitantly, inhibition of the PI3K/Akt pathway. We have reported that rituximab sensitizes B-NHL cells to apoptosis by various chemotherapeutic drugs and demonstrated that inhibition of the PI3K/Akt pathway by various inhibitors mimics rituximab in the sensitization of the tumor cells to apoptosis by chemotherapeutic drugs (Suzuki et al., Oncogene 26:6184, 2007). The role of PTEN induction by rituximab in the sensitization of resistanr B-NHL cells to drug-apoptosis was demonstrated in cells pre-treated with rituximab (to induce PTEN) and then transfected with PTEN siRNA. The transfected cells were resistant to drug-induced apoptosis compared to the control siRNA treated cells. Altogether, the above findings demonstrate that rituximab-induced inhibition of the PI3K/Akt pathway is due, in part, to the induction of PTEN through rituximab-induced inhibition of the PTEN repressors Snail and YY1, downstream of NF-κB. Thus, the induction of PTEN by rituximab plays a major role in the reversal of tumor cell resistance to chemotherapeutic drugs. Further, the findings reveal that the dysregulated PI3K/Akt/NF-κB/Snail/YY1/PTEN loop in B-NHL cells can be interfered by rituximab. This interference leads to the inhibition of cell survival and reversal of resistance through sensitization to drugs. We propose that the gene products in this loop are potential novel therapeutic targets in the treatment of lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Detection of antigens of the mouse mammary tumor (MTV) and murine leukemia virus (MuLV) in cells of cultures derived from mammary tumors of mice of several strains

Virology ◽

10.1016/0042-6822(71)90347-3 ◽

1971 ◽

Vol 45 (2) ◽

pp. 470-483 ◽

Cited By ~ 33

Author(s):

J. Hilgers ◽

W.Clydell Williams ◽

B. Myers ◽

L. Dmochowski

Keyword(s):

Mammary Tumor ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Mammary Tumors ◽

Mouse Mammary Tumor ◽

Murine Leukemia ◽

In Cells

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Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100063792 ◽

1969 ◽

Vol 27 ◽

pp. 376-377

Author(s):

A. M. Watrach

Keyword(s):

Tissue Culture ◽

Small Proportion ◽

Chicken Embryo ◽

Culture Cells ◽

Tissue Culture Cells ◽

Morphologic Characteristics ◽

Infectious Laryngotracheitis ◽

Laryngotracheitis Virus ◽

In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

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Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066796 ◽

1971 ◽

Vol 29 ◽

pp. 548-549

Author(s):

Awtar Krishan ◽

Dora Hsu

Keyword(s):

Granular Material ◽

Rna Synthesis ◽

Culture Medium ◽

Actinomycin D ◽

Metabolic Inhibitors ◽

Protein And Rna Synthesis ◽

Apparent Reduction ◽

Plant Alkaloids ◽

In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

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Great Increase of Numbers of Virus Particles by Irradiating C3H Mouse Mammary Tumors Transplanted into Male Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094620 ◽

1972 ◽

Vol 30 ◽

pp. 272-273

Author(s):

P. J. Melnick ◽

J. W. Cha ◽

E. Samouhos

Keyword(s):

Subcutaneous Tissue ◽

Mammary Tumors ◽

The Body ◽

X Ray ◽

Virus Particles ◽

Mouse Mammary Tumors ◽

Transplantable Tumors ◽

C3h Mice ◽

Cobalt Radiation ◽

Enzyme Changes

Spontaneous mammary tumors in females of a high tumor strain of C3H mice were cut into small fragments that were Implanted into the subcutaneous tissue of the back of males of the same strain, where they grew as transplantable tumors. When about Cm. In diameter daily fractional radiation was begun, applied to the tumors, the rest of the body being shielded by a lead shield. Two groups were treated with 150 and 200 r X-ray dally, of half value layer 0.6mm. copper; a third group was treated with 500 r cobalt radiation dally. The primary purpose was to examine the enzyme changes during radiation, with histochemlcal technics.

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Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162545 ◽

1996 ◽

Vol 54 ◽

pp. 16-17

Author(s):

J. R. Hully ◽

K. R. Luehrsen ◽

K. Aoyagi ◽

C. Shoemaker ◽

R. Abramson

Keyword(s):

Nucleic Acids ◽

Viral Infections ◽

Anatomical Location ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Cellular Source ◽

Gene Transcripts ◽

Initial Description ◽

In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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Absence of temporal ordering of apoptotic features in heat-shock treated leukemia and lymphoma cell lines

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166257 ◽

1996 ◽

Vol 54 ◽

pp. 758-759

Author(s):

D. W. Fairbain ◽

M.D. Standing ◽

K.L. O'Neill

Keyword(s):

Heat Shock ◽

Cell Lines ◽

Chromatin Condensation ◽

Membrane Integrity ◽

Resistant Cell ◽

Membrane Blebbing ◽

Late Loss ◽

Induced Apoptosis ◽

Dying Cells ◽

In Cells

Apoptosis is a genetically defined response to physiological stimuli that results in cellular suicide. Features common to apoptotic cells include chromatin condensation, oligonucleosomal DNA fragmentation, membrane blebbing, nuclear destruction, and late loss of ability to exclude vital dyes. These characteristics contrast markedly from pathological necrosis, in which membrane integrity loss is demonstrated early, and other features of apoptosis, which allow a non-inflammatory removal of dead and dying cells, are absent. Using heat shock-induced apoptosis as a model for examining stress response in cells, we undertook to categorize a variety of human leukemias and lymphomas with regard to their response to heat shock. We were also interested in determining whether a common temporal order was followed in cells dying by apoptosis. In addition, based on our previous results, we investigated whether increasing heat load resulted in increased apoptosis, with particular interest in relatively resistant cell lines, or whether the mode of death changed from apoptosis to necrosis.

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Umatilla Virus Association with Fibrils and Tubules in Cultured Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002063 ◽

1980 ◽

Vol 38 ◽

pp. 476-477

Author(s):

Neil M. Foster ◽

Ruth D. Breckon

Keyword(s):

Bluetongue Virus ◽

Linear Array ◽

Cultured Cells ◽

Intimate Association ◽

Infected Cells ◽

Dark Staining ◽

In Cells

Macrotubules have been described1 in cells infected with Umatilla virus (UMAV), an orbivirus for which bluetongue virus (BTV) is the protype. Macrotubules, often in linear array, were observed in the cytoplasm and in intimate association with viroplasms of infected cells. Macrotubules had outside and inside diameters of 20 and 15 nm and many had dark-staining centers with diameters similar to the interiors of the tubules. UMAV was 60 nm and the RNA core was 30 nm in diameter. This report describes the association of UMAV with macrotubules and two types of microtubules.

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