Jim McBride

Deleted in oral cancer-1 (doc-1), a novel oral tumor suppressor gene.

We have identified, isolated, and par‐tially characterized doc‐1, a novel cDNA sequence whose activity is consistent with a suppressor of hamster oral carcinogenesis. Doc‐1 is an evolutionarily conserved gene exhibiting loss of heterozygosity and marked reduction in expression in malignant hamster oral keratinocytes. The full‐length doc‐1 cDNA encodes an 87 amino acid product that shows a significant homology to one of the seven novel genes induced in mouse fibroblasts by TNF‐α. Transfection of the full‐length doc‐1 cDNA into malignant hamster oral keratinocytes alters the behavior of the recipients in terms of morphology, growth rate, and anchorage‐independent growth, suggesting reversion of transformation phenotypes. We propose that doc‐1 is a novel tumor suppressor gene in oral cancer development.—Todd, R., McBride, J., Tsuji, T., Donoff, R. B., Nagai, M., Chou, M. Y., Chiang, T., Wong, D. T. W. Deleted in oral cancer‐1 (doc‐1), a novel oral tumor suppressor gene. FASEB J. 9, 1362‐1370(1995)

p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein.

ABSTRACT Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12DOC-1 associates with CDK2. More specifically, p12DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12DOC-1transfectants (↑ G1 and ↓ S). The p12DOC-1-mediated decrease of CDK2 was prevented if the p12DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin β-lactone, suggesting that p12DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12DOC-1-mediated, CDK2-associated cell cycle phenotypes. These data support p12DOC-1 as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.

p12DOC-1, a growth suppressor, associates with DNA polymerase α/primase

p12DOC‐1 is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12DOC1 in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12DOC1 associates with DNA polymerase α/primase (pol‐α: primase) in vitro and in cells. The pol‐α:primase binding domain in p12DOC‐1 is mapped to the amino‐terminal six amino acid (MSYKPN). The biological effect of p12DOC1 on pol‐α:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12DOC‐1 suppresses DNA rep lication, leveling at —50%. Similar results were obtained using the M13 single‐stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12DOC1 affects the initiation step, not the elongation phase. The p12DOC1 suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol‐α:primase or by its effect on cyclin‐dependent kinase 2 (CDK2), a recently identified p12DOC‐1‐associated protein known to stimulate DNA replication by phosphorylating pol‐α:primase. p12DOC1 suppresses CDK2‐mediated phosphorylation of pol‐α:primase. These data support a role of p12DOC1 as a regulator of DNA replication by direct inhibition of pol‐α:primase or by negatively regulating the CDK2‐mediated phosphorylation of pol‐α:primase.—Matsuo, K., Shintani, S., Tsuji, T., Nagata, E., Lerman, M., McBride, J., Nakahara, Y., Ohyama, H., Todd, R., Wong, D. T. W. p12DOC‐1, a growth suppressor, associates with DNA polymerase α/primase. FASEB J. 14, 1318–1324 (2000)

doc-1--mediated apoptosis in malignant hamster oral keratinocytes.

PURPOSE Cell cycle mediators involved in inducing apoptosis are frequently deregulated during carcinogenesis. Deleted in oral cancer-1 (doc-1) is an S-phase regulator that is inactivated during oral carcinogenesis. Transfection of doc-1 into malignant oral keratinocytes leads to increased cell loss. It is hypothesized that ectopic expression of doc-1 in hamster oral cancer cells induces apoptosis. MATERIALS AND METHODS Malignant hamster oral keratinocytes (wt-HCPC-1), which lack measurable doc-1 mRNA and protein, were previously transfected with either a CMV-doc-1 expression vector construct (doc-HCPC-1) or the parental control vector pcDNA3 (cv-HCPC-1). A trypan blue exclusion assay was performed to examine cell death in the parental or wild-type HCPC-1 keratinocytes, HCPC-1 transfected with the parental pcDNA3 vector, and the doc-1 transfected HCPC-1 cells. To examine whether ectopic expression of doc-1 mediates gross cellular changes consistent with apoptosis, toluidine blue-safranin differential staining and the quantitative fluorescent microscopy assays were performed. To identify early apoptotic cytochemical changes observed in the cell membrane and nucleus, annexin V/propidium iodide (PI) fluorescence-activated cell sorter (FACS) analysis and the terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL) assay were performed. RESULTS Doc-HCPC-1 showed elevated numbers of dead cells over wt-HCPC-1 and cv-HCPC-1 in the trypan blue exclusion assay. Toluidine blue-safranin staining and quantitative fluorescent microscopy showed significant morphologic changes in the doc-1 transfectants consistent with apoptosis (P < .05). TUNEL assays (P < .05) and annexin V/PI FACS analysis (P < .05) also showed early cytochemical changes in the doc-HCPC-1 transfectants, confirming that ectopic expression of doc-1 induces apoptosis. CONCLUSIONS These data suggest that doc-1 induces apoptosis in malignant hamster oral keratinocytes. It is hypothesized that doc-1 is a mediator of apoptosis that is inactivated during hamster oral carcinogenesis.

Reduction of ornithine decarboxylase antizyme (ODC-Az) level in the 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis model.

Ornithine decarboxylase (ODC) activity is elevated in and necessary for oral carcinogenesis, but the mechanism for its deregulation is unclear. Using subtractive hybridization, a 1029 bp full-length cDNA encoding a 222 amino acid open reading frame has been isolated from normal hamster oral keratinocytes. The hamster cDNA is homologous to the human, mouse and rat ornithine decarboxylase antizyme gene (ODC-Az). The hamster ODC-Az gene demonstrated a restriction fragment length polymorphism (RFLP) upon Southern blot analysis comparing normal and tumor hamster genomic DNA. Northern blot analysis revealed that normal hamster oral keratinocytes express readily detectable level of ODC-Az mRNA. Malignant oral keratinocytes demonstrate reduced expression of the ODC-Az mRNA. In contrast, malignant hamster oral keratinocytes have elevated ODC mRNA levels and lengthened ODC protein half-life when compared to the normal counterparts. This was corroborated by direct measurement of ODC enzymatic activity. These data support the hypothesis that the reduced and/or loss of expression and function of the ODC-Az gene is an important event for the early de-regulation of cellular proliferation during oral tumor development.

IL-1β and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1β (IL-1β) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1β and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10-5 M norepinephrine at 37°C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1β and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1β ( P < 0.03) and IL-6 ( P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1β and IL-6 were significantly higher ( P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1β and IL-6 and release these cytokines from their granules after α- and β-adrenergic stimulation.

Age-related Alterations in IL-1β, TNF-α, and IL-6 Concentrations in Parotid Acinar Cells from BALB/c and Non-obese Diabetic Mice

IL-1β, TNF-α, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sjögrens syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3∊, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p < 0.003) increase in IL-1β and a large significant decrease (p < 0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1β and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:1033–1041, 2000)

Optimized conditions for gene transfection into the human eosinophilic cell line EoL-1 by electroporation

Eosinophils are emerging as an increasingly important cell in the immunoregulatory network of normal and pathological processes. No studies has yet described optimized experimental strategies to transfect DNA into human eosinophils. Using a frequently employed in vitro model of human eosinophil, the EoL-1 cells, we now described the optimal transfection of DNA into these cells by electroporation. Our results indicate that electroporation can efficiently and reproducibly transfect DNA into EoL-1 cells. Optimal electroporation conditions consist of the use of 1 X RPMI medium 1640 with 10% FBS, voltage setting at 275 V, 1150 microF capacitance, 40 mg of DNA and 4.0 X 10(7) cells/ml per electroporation in a total volume of 0.5 ml in 0.4 cm gap cuvettes. These conditions may be a useful protocol for transfecting eosinophil cell lines.

Sequential cytogenetic alterations in hamster oral keratinocytes during DMBA-induced oral carcinogenesis.

Using the hamster cheek pouch oral cancer model, we have performed a comprehensive analysis of the cytogenetic changes in hamster oral keratinocytes during 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. Tumour induction in the hamster cheek pouch required repeated application of the carcinogen for 14 weeks. We have found that this hamster oral cancer model to be suitable for cytogenetic studies. Unlike human oral cancers where chromosome breaks have been shown, this is only infrequently observed in DMBA-treated hamster oral keratinocytes. Of importance is the finding that at the beginning of the second week of DMBA treatment, there is a significant increase of karyotypes demonstrating tetraploid or near-tetraploidy. We propose that the significant increase in hamster oral keratinocytes exhibiting tetraploidy be further evaluated as a marker of premalignancy/malignancy.