Treatment of murine cutaneous melanoma with near infrared light.

Treatment of cutaneous melanoma (M-3 and B16-F10 implanted in mice) with rapidly-scanned, tightly-focused near infrared light elicits selective destruction of tumor tissue. A single laser treatment yielded complete eradication in >90% of B16-F10 tumors with thicknesses of approximately 3 mm; amelanotic M-3 tumors proved less responsive (ca 25% clearance rate). In addition to local tumor destruction, laser treatment of B16-F10 tumors in immunocompetent mice stimulated enhanced cytokine levels (interleukin-2 and interleukin-10) within treated tumor tissues and rejection of tumor cells upon a subsequent challenge dose. Such an antitumor immune response may lead to improved outcomes at both the treatment site and at sites of distant metastasis.

Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein.

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.

Fas-Fas ligand-mediated apoptosis within aqueous during idiopathic acute anterior uveitis.

PURPOSE: Despite ocular immune privilege, (auto)immune-mediated acute anterior uveitis (AAU) is relatively common. However, although relapses of AAU are usually self-limiting, possible regulatory mechanisms remain undefined in humans. Experimentally, Fas-Ligand (FasL)-mediated apoptosis of Fas+ inflammatory cells contributes to the immune privilege within the anterior chamber and provides an explanation for the success of corneal allograft transplantation. Therefore, whether such mechanisms regulate the immune response in AAU was investigated. METHODS: Aqueous and peripheral blood samples from consecutive patients presenting with idiopathic AAU were obtained with consent. Leukocytic phenotype was analyzed by flow cytometry, and apoptosis was determined by both flow cytometry and TdT-dUTP terminal nick-end labeling analysis. Presence of soluble Fas and FasL was determined by western blot analysis and enzyme-linked immunosorbent assay and compared with control aqueous from patients undergoing cataract surgery. The ability of the aqueous to induce apoptosis in a Fas+ Jurkat cell line was also determined. RESULTS: During AAU aqueous-infiltrating Fas+ cells included CD3+ T cells and granulocytes, whereas FasL+ cells comprised predominantly of non-CD3+ T cells. Higher levels of functional soluble FasL were found in aqueous of AAU patients than in normal aqueous, capable of inducing apoptosis in 68.9% +/- 7.6% of Fas+ lymphoid cells. Compared with peripheral blood, the CD4+ T cells infiltrate within aqueous showed significantly increased CD69 and CD25(IL-2r) expression. Flow cytometric analysis of aqueous showed that 9.32% +/- 1.2% of infiltrating non-granulocyte CD45+ cells were apoptotic, confirmed as T cells on subsequent three-color flow cytometric analysis. CONCLUSIONS: Taken together with published experimental data, the present study provides evidence for FasL-mediated apoptotic cell death contributing to the local immune regulation of ocular inflammatory disease and provides a mechanism to account for the self-limiting clinical course of AAU.

Immunosuppressive treatment of choroidal neovascularization associated with endogenous posterior uveitis.

OBJECTIVE: To determine whether resolution of choroidal neovascularization (CNV), a recognized sight-threatening complication of endogenous posterior uveitis, and maintenance of vision could be achieved with immunosuppression. PATIENTS AND METHODS: Fourteen patients (17 eyes) with CNV associated with endogenous posterior uveitis were enrolled in an open study. Ages ranged from 5 to 51 years. Three eyes had extrafoveal CNV, 6 juxtafoveal, and 8 subfoveal. Three patients were treated with a single course of oral corticosteroids, 2 had additional cyclosporine for up to 2 years, and 9 continued to receive a low-dose regimen of a combination of immunosuppressive drugs. RESULTS: After a median follow-up of 15 months (range, 7 months to 6 1/2 years), 9 of 17 eyes had an improvement in visual acuity; 6 remained within 1 Snellen line of initial visual acuity, and 2 had lost 2 Snellen lines. Angiographically, CNV resolved in 13 eyes, resolved then recurred in 3, and improved but persisted in 4. CONCLUSION: These results support a role for immunosuppressive therapy in the treatment of CNV associated with endogenous posterior uveitis.

Xenoestrogens significantly enhance risk for breast cancer during growth and adolescence.

Breast cancer is one of the most common forms of cancer observed in women, and endogenous estrogen is thought to play a major role in its development. Because of this, any conditions or exposures which enhance estrogenic responses would result in an increased risk for breast cancer. The role of xenoestrogenic compounds, such as DDT, in the etiology of breast cancer is still very controversial. In the following paper we discuss recently-published observations by ourselves and others which indicate that xenoestrogens may play a significant role in the development of breast cancer. Specifically, we hypothesize that during periods of high growth rates and during breast development the sensitivity of breast cells to estrogenic compounds is sufficiently great for xenoestrogens to significantly enhance risk for breast cancer.

Interactions of phosducin with the subunits of G-proteins. Binding to the alpha as well as the betagamma subunits.

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein betagamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha and the beta subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha than for beta subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alphao was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein betagamma subunit complexes as well as purified alpha subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao), and 200 nM (alphat). In functional experiments, the steady state GTPase activity of isolated alphao was inhibited by phosducin by approximately 60% with an IC50 value of approximately 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approximately 90% with an IC50 value of approximately 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alphao as measured by single-turnover assays, but it inhibited the release of GDP from alphao; the rate constant of GDP release was decreased approximately 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approximately 100 nM. These data suggest that phosducin binds with high affinity to G-protein betagamma subunits and with lower affinity to G-protein alpha subunits. We propose that the alpha subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the betagamma subunit complex alone.

Simultaneous two-photon activation of type-I photodynamic therapy agents.

The excitation and emission properties of several psoralen derivatives are compared using conventional single-photon excitation and simultaneous two-photon excitation (TPE). Two-photon excitation is effected using the output of a mode-locked titanium: sapphire laser, the near infrared output of which is used to promote nonresonant TPE directly. Specifically, the excitation spectra and excited-state properties of 8-methoxypsoralen and 4'-aminomethyl-4,5,8-trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two-photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two-photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamaging near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage.

Estrogenic and DNA-damaging activity of Red No. 3 in human breast cancer cells.

Exposure to pesticides, dyes, and pollutants that mimic the growth promoting effects of estrogen may cause breast cancer. The pesticide DDT and the food colorant Red No. 3 were found to increase the growth of HTB 133 but not estrogen receptor (ER) negative human breast cells (HTB 125) or rat liver epithelial cells (RLE). Red No. 3, beta-estradiol, and DDT increase ER site-specific DNA binding to the estrogen response element in HTB 133 cells and increase cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells. Site-specific DNA binding by p53 in RLE, HTB 125, HTB 133, and MCF-7 cells was increased when they were treated with Red No. 3, which suggests that cellular DNA was damaged by this colorant. Red No. 3 increased binding of the ER from MCF-7 cells to the estrogen-responsive element. Consumption of Red No. 3, which has estrogenlike growth stimulatory properties and may be genotoxic, could be a significant risk factor in human breast carcinogenesis.

Dietary estrogens stimulate human breast cells to enter the cell cycle.

It has been suggested that dietary estrogens neutralize the effect of synthetic chemicals that mimic the effects of estrogen (i.e., xenoestrogens, environmental estrogens). Genistein, a dietary estrogen, inhibits the growth of breast cancer cells at high doses but additional studies have suggested that at low doses, genistein stimulates proliferation of breast cancer cells. Therefore, if dietary estrogens are estrogenic at low doses, one would predict that they stimulate estrogen-receptor positive breast cancer cells to enter the cell cycle. Genistein and the fungal toxin zearalenone were found to increase the activity of cyclin dependent kinase 2 (Cdk2) and cyclin D1 synthesis and stimulate the hyperphosphorylation of the retinoblastoma susceptibility gene product pRb105 in MCF-7 cells. The steroidal antiestrogen ICI 182,780 suppressed dietary estrogen-mediated activation of Cdk2. Dietary estrogens not only failed to suppress DDT-induced Cdk2 activity, but were found to slightly increase enzyme activity. Both zearalenone and genistein were found to stimulate the expression of a luciferase reporter gene under the control of an estrogen response element in MVLN cells. Our findings are consistent with a conclusion that dietary estrogens at low concentrations do not act as antiestrogens, but act like DDT and estradiol to stimulate human breast cancer cells to enter the cell cycle.

DDT mimicks estradiol stimulation of breast cancer cells to enter the cell cycle.

Estrogens play a critical role in the etiology of found breast cancer. Estradiol promotes the growth of breast cancer cells in vivo and in vitro. Exogenous estrogens in both the environment and in the human diet increase the growth of breast cancer cells in vitro. A role for xenoestrogens in breast cancer etiology has been proposed but remains controversial. We examined the effects of the xenoestrogenic pesticide 1,1,1-trichloro-2,2-bis(chlorophenyl)ethane (DDT) on estrogen-receptor (ER)-positive MCF-7 and T-47D human breast cancer cells as well as on ER-negative HS 578Bst breast cancer cells and rat liver cells. Estradiol and DDT were found to increase the growth of MCF-7 cells in the presence of insulin. The activity of cyclin-dependent kinase (Cdk)2 increased in growth-arrested T-47D and MCF-7 cells treated with beta-estradiol or DDT. The steroidal antiestrogen ICI 182,780 prevented both growth and Cdk2 activation induced by estradiol or DDT. Increased phosphorylation of Cdk2 and the retinoblastoma protein (pRb1O5) was observed in ER-positive cells treated with DDT or estradiol. Cdk2 activity was not affected by DDT or estradiol in ER-negative HS 578Bst breast cancer cells or in rat liver epithelial cells. Cyclin D1 protein synthesis was increased by DDT and estradiol in MCF-7 cells. DDT and estradiol-induced ER-dependent transcriptional activation of estrogen response elements (EREs) in stably transfected MVLN cells, and ERE activation by low doses of DDT was increased by insulin. These findings suggest that DDT can stimulate breast cancer cells to enter into the cell cycle by directly affecting key regulatory elements. The relative potency of DDT in inducing cell-cycle progression appears to be only 100-300 times less than that of estradiol when measured in the presence of insulin. Therefore, the cancer risks associated with DDT exposure may be greater than first thought, especially when additional mitogenic stimuli are present.

Identification of a C-terminal binding site for G-protein betagamma-subunits in phosducin-like protein.

Phosducin-like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G-protein betagamma-subunit (G betagamma)-mediated signaling, with an affinity about 5-fold lower than that of phosducin. The G betagamma binding site of phosducin has been suggested to be contained in its N-terminus. A region corresponding to this N-terminus is lacking in PhLP, suggesting that PhLP must utilize a different mode of G betagamma binding. To map the G betagamma binding site in PhLP, a series of deletion mutants were constructed, expressed in E. coli as glutathione S-transferase (GST) fusion proteins, and the purified fusion proteins were examined for their ability to attenuate G(o) GTPase activity. Progressive N-terminal truncations of PhLP caused only minor reductions in potency, whereas the complementary N-terminal PhLP fragments turned out to be inactive. We further identified a short C-terminal segment comprising residues 168 to 195 that inhibited G0 GTPase activity similar in efficacy and potency to full-length PhLP. This C-terminal fragment was also capable of antagonizing a second G betagamma-mediated function, the enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with G betagamma via a short C-terminal binding site which is distinct from that identified previously in phosducin.

Carcinogenic potential of benzene and toluene when evaluated using cyclin-dependent kinase activation and p53-DNA binding.

Benzene is carcinogenic, whereas toluene is thought to have little carcinogenic potential. Benzene and toluene were found to activate cyclin-dependent kinase 2 in rat liver epithelial (RLE) and HL60 cells. pRb105 was hyperphosphorylated in RLE cells treated with either solvent. Kinase activation and subsequent hyperphosphorylation of pRb105 and p53 by benzene or toluene may be responsible for their growth promotional effects, but it does not account for increased potential of benzene to induce cancer. Therefore, we examined the ability of these solvents to increase p53-DNA site-specific binding in RLE cells. Benzene increased p53-DNA site-specific DNA binding in RLE cells compared to control levels or the effects of toluene. Increased p53-DNA site-specific binding by benzene may be caused by damage to cellular DNA. If so, although both solvents appear to have promotional activity, the increased potential of benzene to damage DNA may be responsible to the difference in the ability of benzene to cause cancer.

Effects of 60-Hz fields, estradiol and xenoestrogens on human breast cancer cells.

If exposure to xenoestrogens or electromagnetic fields (EMFs) such as 60 Hz contributes to the etiology of breast cancer, it is likely that they must stimulate the growth of breast cells, damage genetic material or enhance the effects of other mitogenic or mutagenic agents (co-promotion). Therefore, the ability of xenoestrogens or exposure to 60-Hz fields to stimulate the entry of growth-arrested human breast cancer cells into the cell cycle was determined using cyclin-dependent kinase 2 (Cdk2) activity, synthesis of cyclin D1 and cdc2 activity. Exposure of estrogen receptor-positive MCF-7 or T-47D cells to estrogen and xenoestrogens (DDT and Red No. 3) increased Cdk2 and cyclin B1-cdc2 activity and cyclin D1 synthesis. Exposure of breast cancer cells to 12 mG or 1 or 9 G electromagnetic fields at 60 Hz failed to stimulate Cdk2 or cyclin B1-cdc2 activity or cyclin D1 synthesis. Simultaneous co-exposure of cells to 60-Hz fields and chemical promoters did not enhance Cdk2 activation above the levels produced by the chemical promoter alone. Estrogen and xenoestrogens also stimulated binding of the estrogen receptor to the estrogen receptor element but the EMF did not. Phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of p53 and pRb1O5 in MCF-7 cells, but EMF exposure had no effect. DNA-damaging chemotherapeutic agents and Red Dye No. 3 were found to increase p53 site-specific DNA binding in breast cancer cells, but EMF exposure did not. Differential display analysis failed to detect any effect of EMF exposure on gene expression in MCF-7 cells, whereas the effects of estradiol were detected. These studies suggest that estrogen and xenoestrogens stimulate growth-arrested breast cancer cells to enter the growth cycle, but EMF exposure does not. Site-specific p53-DNA binding was increased in MC F-7 cells treated with DNA-damaging agents, but not by EMF exposure. EMF exposure does not appear to act as a promoter or DNA-damaging agent for human breast cancer cells in vitro.

Phenotypic and genotypic analysis of rat liver epithelial cells infected with retroviral shuttle vectors.

Rat liver epithelial cells (RLE) are suspected to be pluripotent hepatic stem cells that give rise to a diverse variety of liver tumors. The molecular events responsible for transformation of these cells and the diversity of the tumor phenotypes remains to be fully elucidated. We examined the genotype and phenotype of RLE cells infected with retroviral shuttle vectors carrying a neomycin resistance (neor) Ha-ras or a lacZ gene. WBneoIII, WBrasIII and WBlacZ cell lines were examined for evidence of a transformed phenotype by comparing their behavior with the parental strain (WB-344) and with WBneo-C-II and WBrasII cells. Confluent cultures of WBneo-C-II and WBrasII cells were found to contain significantly higher numbers of total cells than the other cell lines. The growth rate of WBneo-C-II and WBrasII cells were faster than that of the parental cell line. Addition of epidermal growth factor (EGF) to the medium was found to stimulate the growth rate of WBneo-C-II cells and to induce anchorage independent growth (AIG). No cell line produced tumors in nude mice (nu/nu) except WBrasII cells. Radioimmunoprecipitation studies and sequencing of the p53 exons 5-8 indicate WBneo-C-II, and WBrasII cells produce a mutant p53. Northern blot analysis showed an increased expression of c-myc mRNA in WBneo-C-II and WBrasII cells. These results demonstrate that alterations in critical growth and differentiation controlling genes have occurred in WBrasII cells which may, independent of or in conjunction with ras insertion, cause the transformed phenotype.

Wild type p53 reduces the size of tumors caused by bovine leukemia virus-infected cells.

Bat lung (BAT(2)CL6) cells infected with bovine leukemia virus (BLV) cause malignant tumors in nude mice that after 6 weeks subcutaneous growth, have an average volume of 0.3m(3). Uninfected bat lung cells (Tb 1 Lu) produce small benign neoplasms that average 0.003 cm(3). BAT(2)CL6 cells were transfected in vitro with expression vectors that produce wild type human or mutant p53. Production of human p53 in transfected BAT(2)CL6 cells was confirmed by immunoprecipitation of p53 and by immunohistochemical staining using anti-human p53 monoclonal antibodies. BAT(2)CL6 cells transfected with wild type p53 produced tumors in nude mice averaging 0.03 cm(3) whereas cells transfected with mutant p53 yielded tumors averaging 0.3cm(3). BAT(2)CL6 cell tumors after 1 week subcutaneous growth were transfected in situ with the wild type p53 gene. At 6 weeks tumor volume of in situ transfected tumors was similar to those resulting from cells transfected in vitro. Histopathologic examination and immunochemical staining of tumors produced in nude mice after wild type p53 treatment showed no significant differences when compared to tumors produced by untreated BAT(2)CL6 cells. Therefore, it is likely that the tumors produced by p53 treated-cells arose from cells that escaped transfection. The reduction of tumor size by restoration of wild type 53 may prove to be a useful therapy for BLV-induced tumors.

A role for trkA nerve growth factor receptors in mammalian ovulation.

Several members of the neurotrophin (NT) family, including nerve growth factor (NGF), NT-3, and NT-4/5, are expressed in the mammalian ovary. As their respective receptor tyrosine kinases are also found in the gland, the possibility exists that NTs act directly on the gonads to exert effects unrelated to their support of the ovarian innervation. We now report that trkA, the NGF receptor tyrosine kinase, is involved in the acute activational process that leads to the first ovulation. The trkA gene becomes transiently expressed in periovulatory follicules at the time of the first preovulatory surge of gonadotropins at puberty; the increase in trkA messenger RNA (mRNA) content is dramatic ( > 100-fold), but transient (approximately 9 h). No such changes in trkB or trkC mRNA were observed; the abundance of these mRNAs, which encode the receptor tyrosine kinase for NT-4/5 and brain-derived neurotrophic factor, and NT-3, respectively, remained at very low levels throughout puberty. In vivo and in vitro experiments demonstrated that the activation of trkA gene expression is brought about by the proestrous discharge of LH. The increase in trkA mRNA levels is mainly localised to cells of the follicular wall and interstitial tissue of the ovary. NGF mRNA abundance also increases at proestrus, with peak values detected about 5 h before ovulation; as in the case of trkA mRNA, NGF mRNA was found in thecal-interstitial cells. Both trkA and NGF protein, detected by immunohistochemistry, were localized to this same ovarian compartment. Interleukin-1 beta (IL-1 beta), a putative mediator of LH action, enhances both trkA and NGF gene expression in ovarian cells, an effect prevented by IL-1ra, a natural IL-1 beta receptor antagonist. Il-1 beta also stimulates PGE2 release, and this effect was inhibited by both NGF antibodies and a trk receptor blocker, NGF antibodies administered in vivo attenuated the increase in ovarian PGE2 synthesis that antedates ovulation. Immunoneutralization of NGF action or pharmacological blockade of trk tyrosine kinase activity targeted to one ovary resulted in the ipsilateral inhibition of ovulation. The remarkably narrow time frame of trkA gene activation at the completion of follicular growth suggests that NGF acting as a neuroendocrinotrophic factor in a developmentally restricted manner contributes to the acute cytodifferentiation process that leads to the first ovulation in mammals.

Increased p53 site-specific DNA binding in cells producing mutant p53.

Chemotherapeutic and DNA-damaging agents were found to increase p53 site-specific DNA binding in human breast and rat liver epithelial WBrasII cells which produce mutant p53. Increased p53 site-specific DNA binding by chemotherapeutic or DNA-damaging agents was also induced in transfected Saos-2 cells producing wild type or transforming mutant p53. Therefore, exposure of cells containing a transforming p53 mutant to chemotherapeutic or DNA-damaging agents may potentially enhance their transformation state and tumorigenic potential.

Ultrasonographic investigation of optic neuritis.

A pilot echographic study was conducted to determine the incidence and severity of optic nerve swelling in acute 'idiopathic' optic neuritis and to examine cerebrospinal fluid dynamics in the subarachnoid space, employing the method of standardised echography and the '30 degrees test'. An attempt was made to correlate the degree of nerve swelling with the initial visual loss and with the rate and extent of recovery of vision. The visual function and echographic features of the optic nerve in 27 patients with the diagnosis of acute optic neuritis were assessed with standardised echography. A significant increase in nerve diameter was found in 74% of cases. There was a correlation between nerve swelling and the severity of initial visual loss. The authors conclude that standardised echography is a useful tool in the diagnosis of optic neuritis and may play a role in predicting the visual outcome.

Stabilization of the p53 gene product in two bovine leukemia virus infected cell lines.

Fetal lamb kidney cells (FLK) and bat lung (BAT2CL6) cells that continuously produce bovine leukemia virus (BLV) were found to cause malignant tumors in nude mice. Uninfected bat lung cells (Tb 1 Lu) produced a small benign neoplasm. Pulse chase studies showed that the p53 gene product in BAT2CL6 cells was more stable compared with p53 in Tb 1 Lu cells. Mono-clonal antibody studies suggested that a mutant form of the p53 protein was produced in BLV-infected cells. Heteroduplex mapping studies of the p53 gene from BLV-infected cells also suggested that a mutation in p53 had occurred. Stabilization of the p53 gene product in BLV-infected cells may contribute to the progression of tumor virulence.

Hyperphosphorylation of p53 induced by benzene, toluene, and chloroform.

Phorbol 12-myristate, 13-acetate (PMA) is a known protein kinase C activator (PKC); benzene, chloroform, and toluene have also been reported to be PKC activators. We examined the effects of these three solvents on the phosphorylation of p53 in treated cells. Hyperphosphorylated p53 was found when p53 was immunoprecipitated from rat liver epithelial cell extracts treated with any of the solvents or PMA. The solvents also resulted in hyper-phosphorylation of human p53 produced by transfection of Saos-2 cells with a eucaryotic expression vector. Increased phosphorylation of p53 induced by the solvents was also observed through in vitro assays. Hyperphosphorylation of p53 may be involved in tumor promotion by benzene, toluene and chloroform.