James S. Hatfield

Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437.

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes ∼130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.

Tumor cell-platelet interactions in vitro and their relationship to in vivo arrest of hematogenously circulating tumor cells

Aggregation of rat platelets was induced in vitro by homologous rat Walker 256 carcinosarcoma cells and the extent of tumor cell-platelet interactions examined ultrastructurally. By 30s there was surface contact between unstimulated platelets and tumor cell microvilli. By midphase aggregation (2–3 min) tumor cells became enmeshed within expanding platelet aggregates. Tumor cell microvilli and platelet pseudopodia interdigitated as aggregation progressed. During the later stages of aggregation (6–10 min) tumor cells formed large processes which penetrated deep into the aggregate. Platelet activation (i.e. degranulation) occurred in gradient fashion and was concentrated near tumor cell membrane sites involved in process formation. At these later stages tumor cells near the aggregate periphery were found to have engulfed platelets or platelet fragments.Tumor cell-platelet interactions in the pulmonary microvasculature were also studied in vivo following injection of murine Lewis lung carcinoma, 16C mammary adenocarcinoma, and B16 amelanotic melanoma tumor cells into the tail vein. Platelets demonstrated a biphasic association with arrested tumor cells with peak interactions occurring at 10–30 min and 4–24h. Ultrastructurally, tumor cells exhibited newly formed processes which interdigitated with the platelet aggregate. Such processes formed only in areas of contact with platelets and not in areas of contact with endothelial cells or other blood elements (i.e. erythrocytes, polymorphonuclear leukocytes). Numerous tumor cell mitochondria were concentrated in the areas of greatest platelet-tumor cell process activity. At early time intervals (2–10 min), intravascular platelet degranulation was observed primarily in platelets associated with tumor cell processes. Tumor cells also were found to have engulfed platelet fragments in vivo.

Plasma membrane-associated cysteine proteinases in human and animal tumors

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.

Calcium channel subunits in the mouse cochlea.

Abstract: Messages for subunits of voltage‐gated calcium channels were examined in the cochlea of the CBAJ mouse by PCR analysis. Total RNA was extracted from the auditory organs of 16–18‐day‐old animals. After reverse transcription, resulting cDNA was amplified by PCR with primers targeted to nucleotide sequences corresponding to 12 different calcium channel subunits. PCR products representing subunit gene expression were strongly and consistently amplified for α1C, α1D, α1E, α2δ, β1, β3, and β4 but not for α1A, α1B, α1S, β2, or γ. The chosen primers amplified cochlear cDNA to yield an overall pattern of bands different from that of any tissue studied thus far, in particular with respect to the α2δ and β1 subunits; the α2δ product was found to be significantly shorter than the corresponding brain and skeletal muscle isoforms. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. The results suggest that L‐type and presumptive R‐type calcium channels are expressed in the mammalian cochlea and that the α2δ subunits may be coded by a characteristic splice‐variant mRNA.

αIIbβ3 Integrin expression and function in subpopulations of murine tumors

Abstract Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G 2 M phase of the cell cycle. Both B16a and 3LL cells express αIIbβ3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of αIIbβ3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with αIIbβ3 -specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that αIIbβ3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the αIIbβ3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

Transforming Growth Factor-β Stimulates Intestinal Epithelial Focal Adhesion Kinase Synthesis via Smad- and p38-Dependent Mechanisms

Focal adhesion kinase (FAK) regulates cell migration, proliferation, and apoptosis. FAK protein is reduced at the edge of migrating gut epithelial sheets in vitro, but it has not been characterized in restitutive gut mucosa in vivo. Here we show that FAK and activated phospho-FAK (FAK(397)) immunoreactivity was lower in epithelial cells immediately adjacent to human gastric and colonic ulcers in vivo, but dramatically increased in epithelia near the ulcers, possibly reflecting stimulation by growth factors absent in vitro. Transforming growth factor (TGF)-beta, but not fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor, increased FAK levels in Caco-2 and IEC-6 cells. Epithelial immunoreactivity to TGF-beta and phospho-Smad3 was also higher near the ulcers, varying in parallel with FAK. The TGF-beta receptor antagonist SB431542 completely blocked TGF-beta-induced Smad2/3 and p38 activation in IEC-6 cells. SB431542, the p38 antagonist SB203580, and siRNA-mediated reduction of Smad2 and p38alpha prevented TGF-beta stimulation of both FAK transcription and translation (as measured via a FAK promoter-luciferase construct). FAK(397) levels were directly related to total FAK protein expression. Although gut epithelial motility is associated with direct inhibition of FAK protein adjacent to mucosal wounds, TGF-beta may increase FAK protein near but not bordering mucosal ulcers via Smad2/3 and p38 signals. Our results show that regulation of FAK expression may be as important as FAK phosphorylation in critically influencing gut epithelial cell migration after mucosal injury.

Morphological and biochemical changes in gastric mucosa of aging rats

Although previous data from this laboratory have indicated that aging is associated with increased gastric mucosal proliferative activity, no direct assessment of proliferative potential of the tissue has been made during aging. In order to assess this, and to determine whether changes in mucosal proliferative potential would be reflected in growth of the tissue, we have examined the labeling index (LI), height and morphology of the gastric mucosa in young (4-month-old) and aged (24-month-old) Fischer-344 rats. In addition, tyrosine kinase (Tyr-k) activity and the levels of phosphotyrosine proteins were determined to evaluate their relationship to mucosal proliferative activity. Histologic evaluation revealed a marked atrophy of the mucosal glandular component with 32% reduction in height in aged rats when compared with young animals. In aged rats, there was also a decrease in gland density, resulting in a reduction in the number of epithelial cells of all types with evidence of decreased secretory activity. Despite the occurrence of mucosal atrophy in aged rats, LI in these animals was significantly increased by 28%. This was associated with a parallel rise in mucosal Tyr-k activity, and a two-to threefold increase in the relative concentrations of seven phosphotyrosine membrane proteins with Mr of 120, 105, 90, 60, 55, 48 and 32 kDa. We conclude that (1) although aging is associated with increased gastric mucosal proliferative activity, this does not result in mucosal growth and that (2) Tyr-k and tyrosine phosphorylation of certain proteins play a role in the regulation of gastric mucosal cell proliferation during aging.

Selective expression of serotonin receptor transcripts in the mammalian cochlea and its subdivisions.

Expression of serotonin receptor (5-HTR) mRNA has been determined in the mammalian cochlea and its subdivisions by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Specific primers targeting individual 5-HTRs 1-7 directed amplification of 5-HTR subtypes 1A, 1B, 2B, 2C, 3, 5B, and 6 from mouse cochlea cDNA. No evidence of expression was obtained for 5-HTRs 1D, 2A, 4 (L and S), 5A, and 7. The distribution of receptor mRNA within the cochlea was determined with application of RT-PCR to morphologically defined microdissected subfractions of the rat cochlea. Messages for 5-HTR subtypes 1A, 1B, 2B, and 6 were present in the organ of Corti, lateral wall, and spiral ganglion subfractions. Messages for 5-HTR subtypes 2C, 3 and 5B were found in the spiral ganglion, but not in the organ of Corti or lateral wall fractions. The existence of transcripts for 5-HTRs 1A, 1B, 2B and 6 in the organ of Corti is consistent with a role for these receptors in serotonin-mediated modulation of the mechanosensory signal.

Transactivator of transcription–tagged cell cycle and apoptosis regulatory protein-1 peptides suppress the growth of human breast cancer cells in vitro and in vivo

Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)–tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1–198, 197–454, and 896–1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1–198Y192/F) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1–198Y192/F), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1–198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1–198Y192/F), inhibits growth of human breast cancer cell–derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing. [Mol Cancer Ther 2007;6(5):1661–72]

Expression of adenylyl cyclase type I in cochlear inner hair cells

Expression of calcium/calmodulin-activated adenylyl cyclase type I (ACI) mRNA has been determined in the cochlea and in an organ-of-Corti subdissected tissue fraction by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification products of predicted size were obtained from the mouse cochlea and rat organ of Corti with nucleotide sequences corresponding to respective ACI brain transcripts. In addition, ACI template was detected in a rat inner hair cell cDNA library by PCR. Immunoreactivity to ACI has been localized within the organ of Corti to the inner hair cell, with diaminobenzidine staining found in both the cell body and in the stereocilia. Evidence, thus, has been obtained that both ACI transcript and protein are expressed in the inner hair cell, the primary mechanosensory receptor cell of the cochlea. We hypothesize that ACI is activated by calcium influx through a calcium/calmodulin interaction and that this adenytyl cyclase isoform may have a role in modulation of receptoneural afferent transmission and/or mechanosensory transduction in the cochlea.