The IGF-1/IGF-1R signaling axis in the skin: a new role for the dermis in aging-associated skin cancer.

The appropriate response of human keratinocytes to ultraviolet-B (UVB) is dependent on the activation status of the insulin-like growth factor 1 (IGF-1) receptor. Keratinocytes grown in conditions in which the IGF-1 receptor is inactive inappropriately replicate in the presence of UVB-induced DNA damage. In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts. We now show that the IGF-1 produced by human fibroblasts is essential for the appropriate UVB response of keratinocytes. Furthermore, the expression of IGF-1 is silenced in senescent fibroblasts in vitro. Using quantitative reverse transcriptase-PCR and immunohistochemisty, we can show that IGF-1 expression is also silenced in geriatric dermis in vivo. The diminished IGF-1 expression in geriatric skin correlates with an inappropriate UVB response in geriatric volunteers. Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1. These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

Decreased incidence of nonmelanoma skin cancer in patients with type 2 diabetes mellitus using insulin: a pilot study.

BACKGROUND: In order to prevent the propagation of genetic mutations, human keratinocytes irradiated with ultraviolet (UV) B light in vitro undergo premature stress-induced senescence or apoptosis. This response to UVB irradiation is dependent on the functional activation of the insulin-like growth factor-1 receptor (IGF-1R). Based on this in vitro functional data, we hypothesized that the increased serum levels of insulin in patients with type 2 diabetes may activate the IGF-1R in skin and lead to a decreased frequency of skin cancer in these patients. OBJECTIVES: To determine whether the use of insulin by patients with type 2 diabetes correlated with a change in the incidence in nonmelanoma skin cancer (NMSC). METHODS: A historical cohort study identifying the incidence of NMSC following the use of two different pharmacological therapies. The patient population was restricted to caucasians who were at least 50 years old when they began the indicated pharmacological therapy. The first group consisted of 1440 patients who used insulin therapy to treat type 2 diabetes and the second group comprised 4135 patients who used cimetidine to treat their gastrointestinal ailments. An additional group of 6131 patients with diabetes who used noninsulin antidiabetics was added to examine the effect of noninsulin therapies. All patients had regular follow-up visits at the Regenstrief Clinics during the study period between 1980 and 1999. The Regenstrief Clinics is an outpatient facility which serves the general population in Metro-Indianapolis, Indiana, U.S.A. RESULTS: The incidence of NMSC in patients using insulin was significantly lower than in patients using cimetidine (1.25% vs. 2.35%, P < 0.02). The decrease in NMSC in patients with type 2 diabetes correlated specifically with the use of insulin (NMSC incidence insulin-only patients with diabetes: 1.40% vs. those with diabetes using noninsulin therapies: 2.35%, P = 0.11). CONCLUSIONS: Patients using exogenous insulin had a lower risk of developing NMSC and the protective effect of insulin use becomes more distinct with increasing age.

Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells.

The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.

The platelet-activating factor receptor protects epidermal cells from tumor necrosis factor (TNF) alpha and TNF-related apoptosis-inducing ligand-induced apoptosis through an NF-kappa B-dependent process.

A number of chemical mediators can induce human keratinocytes and epidermal-derived carcinomas to undergo apoptosis, or programmed cell death. Recent evidence suggests pro-inflammatory cytokines, such as interleukin-1 beta or transforming growth factor alpha, protects carcinomas from numerous pro-apoptotic stimuli. Platelet-activating factor (1-alkyl-2-acetyl-3-glycerophosphocholine; PAF) is a lipid mediator with pro-inflammatory effects on numerous cell types. Although PAF can be metabolized to other bioactive lipids, the majority of PAF effects occur through activation of a G protein-coupled receptor. Using a model system created by retroviral transduction of the PAF receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we now demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. The PAF-mediated protection was inhibited by PAF-R antagonists, and protection did not occur in PAF-R-negative KB cells. Additionally, we show protection from TNFalpha- or TRAIL-induced apoptosis by PAF-R activation is dependent on the transcription factor nuclear factor (NF)-kappa B, because PAF-R activation-induced NF-kappa B and epidermal cells transduced with a super-repressor form of inhibitor kappa B were not protected by the PAF-R. These studies provide a mechanism whereby the epidermal PAF-R, and possibly other G protein-coupled receptors, can exert anti-apoptotic effects through an NF-kappa B-dependent process.

A system for simultaneous ultraviolet light and electromagnetic field exposure in in vitro experiments.

Ultraviolet light (UV) is a common treatment for skin diseases such as psoriasis, but bears the risk of carcinogenic side effects. We have biological evidence that electromagnetic fields (EMFs) can act additively with UV so that new therapeutic protocols combining UV and EMF might be developed to improve psoriasis phototherapy. In this study we report on a system that allows in vitro experiments testing this hypothesis. For simultaneous exposure of cell cultures to UVB and EMF, we built Merritt coils with an integrated UV exposure system. The coils can be operated in a sham or experimental mode (up to 1.5 mT and 20,000 Hz). Two UV bulbs were fitted inside the coils for UVB doses between 100-1000 J/m2/nm. In the exposure area the EMF is uniform within 0.0038%. For exposure, the cells are cultured in standard culture plates and placed in a specifically designed box. The box holds two plates in a top chamber covered with a Saran Wrap lid (91% UV transmission) so that cells are exposed to UVB and EMFs. The bottom chamber holds two plates, where cells are screened from UVB and only exposed to EMFs. Temperature control is maintained (+/- 1 degree C) by airflow vents on the side of the box and a fan placed 25 cm away from the cell culture box. To maintain sterility within the box the vents are covered with a bacterial filter. The box lid has additional ventilation through two air direction changes to create an additional bacterial barrier similar to that in culture plate lids.

Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes.

Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.

Evidence for involvement of the epidermal platelet-activating factor receptor in ultraviolet-B-radiation-induced interleukin-8 production.

Ultraviolet B radiation has been shown to generate cutaneous inflammation in part through inducing oxidative stress and cytokine production in human keratinocytes. Amongst the proinflammatory cytokines synthesized in response to ultraviolet B radiation is the potent chemoattractant interleukin-8. Though the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and keratinocytes express PAF receptors linked to cytokine biosynthesis, it is not known whether PAF is involved in ultraviolet-B-induced epidermal cell cytokine production. These studies examined the role of the PAF system in ultraviolet-B-induced epidermal cell interleukin-8 biosynthesis using a novel model system created by retroviral-mediated transduction of the PAF-receptor-negative human epidermal cell line KB with the human PAF receptor. Treatment of PAF-receptor-expressing KB cells with the metabolically stable PAF receptor agonist carbamoyl-PAF resulted in increased interleukin-8 mRNA and protein, indicating that activation of the epidermal PAF receptor was linked to interleukin-8 production. Ultraviolet B irradiation of PAF-receptor-expressing KB cells resulted in significant increases in both interleukin-8 mRNA and protein in comparison to ultraviolet-B-treated control KB cells. Pretreatment with PAF receptor antagonists inhibited both carbamoyl-PAF-induced and ultraviolet-B-induced interleukin-8 production in the PAF-receptor-positive cells, but not in control KB cells. Similarly, treatment of the PAF-receptor-expressing primary cultures of human keratinocytes or the human epidermal cell line A-431 with carbamoyl-PAF or ultraviolet B radiation resulted in interleukin-8 production that was partially inhibited by PAF receptor antagonists. These studies suggest that the epidermal PAF receptor may be a pharmacologic target for ultraviolet B radiation in skin and thus may act to augment ultraviolet-B-mediated production of cytokines such as interleukin-8.

Quantitative analysis of UVB-induced apoptosis in human epidermis.

The quantitative measurement of the induction of apoptosis in cells grown in vitro can be accomplished using a variety of proven methods. However, the quantitative assay of apoptosis within an intact tissue is very laborious and the results can be misleading. We have established a method to quantitatively analyze the induction of apoptosis in human epidermis following UVB irradiation. The assay is based on the activation of the apoptotically induced enzyme caspase 3, using a synthetic caspase 3 substrate. The activation of caspase 3 was shown to correlate with the induction of apoptosis in human keratinocytes cultures as a monolayer. We then demonstrated that the activation of caspase 3 could be measured from UVB-irradiated whole skin. The induction of apoptosis was confirmed by cellular morphology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling. Therefore, we concluded that the measurement of caspase 3 specific activity in UVB-irradiated human epidermis was an efficient, inexpensive, and accurate method to quantitate UVB-induced apoptosis in vivo.

Activation of the insulin-like growth factor-1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation.

The ultraviolet B (UVB) component of sunlight causes non-melanoma skin cancers due to the damage it inflicts on genomic DNA. The response of epidermal keratinocytes to sunlight depends on the dose of UVB received and the severity of the damage to the DNA. Mild DNA damage typically induces DNA-repair pathways and cell survival, while severe DNA damage provokes apoptosis. Primary human keratinocytes grown in serum-free media respond in a similar manner to UVB irradiation. However, we observed that keratinocytes are exquisitely more susceptible to UVB-induced apoptosis if the growth medium is depleted of exogenous growth factors. Therefore, an exogenous growth factor could provide protection from UVB-induced apoptosis. We found that the only growth factor that provided protection from UVB-induced apoptosis was insulin and that the protective effect elicited by insulin was not due to binding the insulin receptor but, rather, to activation of the insulin-like growth factor-1 (IGF-1) receptor. Additionally, activation of the IGF-1 receptor in combination with UVB irradiation induced keratinocytes to become post-mitotic. This survival function of the IGF-1 receptor in response to UVB irradiation was influenced by activation of phosphatidylinositol-3 kinase and MAP kinase. Prior to UVB irradiation, insulin or IGF-1 had little to no effect on cell growth or viability. Therefore, activation of the IGF-1 receptor in conjunction with UVB irradiation promotes keratinocyte survival at the expense of cell proliferation.

Subphysiological concentrations of extracellular calcium sensitize normal human keratinocytes to UVB-induced apoptosis.

Normal human keratinocytes are stimulated to proliferate in serum-free medium containing subphysiological concentrations of calcium (0.09 mM, low calcium). In this study, we examined the effect of increased levels of extracellular calcium (2.0 mM, normal calcium) on UVB-induced apoptosis. Apoptosis was assessed by changes in cellular morphology, annexind V-FITC flow cytometry, and the formation of internucleosomal DNA ladders. High doses of UVB induced keratinocytes grown in low calcium medium to undergo apoptosis. In contrast, keratinocytes grown for 72 h in normal calcium medium were completely resistant to UVB-induced apoptosis. No apoptosis was observed even at UVB doses as high as 1200 J/m2. However, despite the lack of UVB-induced cell death, keratinocytes grown in normal calcium medium lost the ability to proliferate following high levels of UVB irradiation. High doses of UVB also increased the expression of the differentiation-specific proteins involucrin and cytokeratin 10 in a dose-dependent manner. In addition, growth in normal calcium medium lowered the UVB-induced stimulation of the p53 protein and altered the normal subcellular localization pattern of p53. UVB irradiation of human keratinocytes grown in normal calcium medium may be inducing further cell differentiation in the absence of overt cell death.

Expression of the platelet-activating factor receptor results in enhanced ultraviolet B radiation-induced apoptosis in a human epidermal cell line.

Recent studies have demonstrated that ultraviolet B radiation (UVB) damages human keratinocytes in part by inducing oxidative stress and cytokine production. Severe UVB damage to the keratinocyte can also result in apoptosis or programmed cell death. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to epidermal cell damage and epidermal cells express PAF receptors, it is not known whether PAF is involved in UVB-induced epidermal cell apoptosis. These studies examined the role of the PAF system in UVB-induced epidermal cell apoptosis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Expression of the PAF-R in KB cells did not affect base-line growth or apoptosis, yet resulted in a decrease in the lag time between treatment of the cells and the induction of apoptosis following irradiation with 400 J/m2 UVB. This effect was inhibited by pretreatment with the PAF-R antagonists WEB 2086 and A-85783, confirming involvement of the PAF-R in this process. At lower doses (100-200 J/m2) of UVB, only KB cells that expressed the PAF-R became apoptotic. Treatment of PAF-R-expressing KB clones with the metabolically stable PAF-R agonist 1-hexadexyl-2-N-methylcarbamoyl-3-glycerophosphocholine (CPAF) alone did not induce apoptosis but augmented the degree of apoptosis observed if CPAF was used in combination with lower doses (200 J/m2) of UVB irradiation. Interestingly, UVB irradiation was found to stimulate PAF synthesis only in PAF-R-expressing KB cell clones. The antioxidants N-acetyl cysteine, 1,1,3,3-tetramethyl-2-thiourea, and vitamin E inhibited both UVB-induced PAF biosynthesis as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB clones, suggesting the possibility that UVB stimulates the production of oxidized lipid species with PAF-R agonistic activity in this model system. Thus, these studies indicate that a component of UVB-induced epidermal cell cytotoxicity can be modulated by PAF-R activation through the production of PAF and PAF-like species.

Ultraviolet B-radiation dose influences the induction of apoptosis and p53 in human keratinocytes.

p53 is a tumor suppressor gene that has been implicated in a number of important cellular processes, including DNA repair and apoptosis. Genomic damage in human keratinocytes caused by ultraviolet B (UVB) irradiation has been shown to induce both apoptosis and p53 expression. We have previously observed that p53 expression in cultured normal human keratinocytes is predominantly perinuclear; however, exposure of cells to UVB radiation induces a major shift of p53 expression to the nucleus. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, internucleosomal DNA ladders and flow cytometry, we correlated observed changes in p53 expression with the induction of apoptosis at low, intermediate and high doses of UVB radiation. High doses of UVB radiation induced cells to undergo apoptosis, whereas UVB radiation at low doses did not induce apoptosis but appeared to stimulate repair of the DNA damage induced by UVB radiation. Intermediate doses of UVB radiation induced a heterogeneous population of cells to undergo either DNA repair or apoptosis. The level of UVB radiation dose also influenced the induced cellular localization of p53. These observed differences in p53 cellular localization correlated with the induction of DNA repair or apoptosis. In cells undergoing apoptosis, p53 protein was found within the blebs of the degenerating nuclei. Our data give support to increasing evidence that p53 may play a role in both the repair of UV-radiation-induced DNA damage and the induction of apoptosis, and may function as a central control checkpoint in response to UVB-radiation-induced DNA damage.

p53-binding proteins in squamous cell carcinoma and normal human keratinocytes.

In squamous cell carcinomas (SCCs), the tumor suppressor protein p53 is frequently overexpressed. The overexpression of p53 is often due to a mutation in the p53 gene; however, increased levels of p53 protein can be observed in the tumors without p53 gene mutations. In normal human keratinocytes, p53 is a multiconformational protein. The different conformations of p53 can be identified by their reactivity with epitope-specific, anti-p53 monoclonal antibodies. This study provides evidence that the different p53 conformations seen in human keratinocytes bind to distinct cellular proteins. Proteins that bind p53 in normal human keratinocytes were compared with p53-binding proteins from cells derived from SCC tumors by immunoprecipitation of [35S]methionine-labeled and 32P(i)-labeled cell lysates using a panel of anti-p53 monoclonal antibodies. In one tumor, the SCC cells contained a protein of Mr 30,000 bound to p53 that was not seen in normal human keratinocytes. Cells derived from a separate SCC did not have the Mr 30,000 protein but did contain two proteins of Mr 15,000 and Mr 16,000, which were not seen in normal human keratinocytes. The immunofluorescent staining pattern of cultured normal human keratinocytes, cells derived from two SCCs, as well as the original tumors from which the cells were derived, was also examined. The immunofluorescent staining of the cells derived from the tumors and the tumors themselves was different from that seen in normal cultured keratinocytes and normal epidermis. These studies suggest that there are alterations in the proteins that bind to p53 in SCCs.

The effect of phosphorylation on the antigenic reactivity of p53 in cultured human keratinocytes.

The detection of p53 in human keratinocytes is dependent on the specific anti-p53 monoclonal antibody that is used. Differences in antibody recognition are postulated to be due to the masking or exposure of particular epitopes in different conformations of p53. This study addresses the role of phosphorylation on p53-epitope accessibility in human keratinocytes. Keratinocytes were treated with the phosphatase inhibitor, okadaic acid, to determine the effect of inhibiting cellular phosphatases on p53 phosphorylation and epitope recognition. These studies suggest there is a correlation between the level of p53 phosphorylation and the antigenic reactivity of certain p53 epitopes in human keratinocytes. We also examined the ability of the catalytic subunits of protein phosphatase 1 and 2A to dephosphorylate p53 derived from human keratinocytes in vitro. These data suggest that PP2A may be the phosphatase that acts on p53 in cultured human keratinocytes.

Distinct conformations of p53 are observed at different stages of keratinocyte differentiation.

p53 is a multifunctional protein that has been shown to inhibit the growth of transformed cells, arrest the cell cycle of normal cells, regulate gene transcription and influence cellular differentiation, apoptosis and senescence. This study was undertaken to determine if the expression of p53 in keratinocytes varied during epidermal differentiation. Fresh frozen-sections of normal human epidermis were subjected to immunofluorescence using a panel of anti-p53 monoclonal antibodies. The monoclonal antibody 122 specifically stained the basal layer of the epidermis. No staining was observed in other cell layers of the epidermis using the 122 antibody. When the 240 antibody was used, p53 was only detected in granular layer cells. No other anti-p53 monoclonal antibody stained normal epidermis. Immunofluorescent analyses of cultured keratinocytes revealed staining patterns that correlated with the staining pattern seen in vivo. Immunoprecipitation assays of the cultured keratinocytes indicated that each monoclonal antibody, with the exception of DO-1, could only detect a fraction of the total p53 present in the cultures. This diversity of reactivity was presumed to be due to the masking and exposing of the various epitopes on p53 through the binding of other proteins. Finally, in cultured human keratinocytes, p53 was found to be a relatively stable protein with a half-life of 5 h.

Repression of the hepatitis B virus enhancer by a cellular factor.

The enhancer element of hepatitis B virus (HBV) regulates the transcription of all HBV mRNA, including the pregenomic mRNA used during replication. This pregenomic mRNA is transcribed from the core gene promoter which is located 500 bp downstream from the HBV enhancer element. To examine the effect of the HBV enhancer on the activity of the core gene promoter, we constructed various plasmids containing different combinations of HBV enhancer and core gene promoter sequences regulating the expression of the chloramphenicol acetyltransferase gene. When the HBV enhancer was positioned immediately adjacent to the core gene promoter, the enhancer increased the activity of the core gene promoter nearly 30-fold. In contrast, the HBV enhancer only modestly stimulated the core gene promoter (less than threefold) at its native position in the HBV genome. This weak HBV enhancer activity was due to a DNA sequence located between the enhancer and the core gene promoter and not due to the increased distance between the enhancer and the core gene promoter. Competition experiments demonstrated that a trans-acting factor(s) bound this sequence and repressed the enhancer. This DNA sequence contains the C/EBP, AP-1 and NF-1 regulatory sites. No inhibition of enhancer activity was observed when only the AP-1 and C/EBP sites were present. Repression of the HBV enhancer was not detected when the NF-1 site was disrupted, indicating that the NF-1 site was involved in the suppression of the HBV enhancer.

A functional hepatitis B virus X protein produced in insect cells.

We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV-1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.

trans-activation of viral enhancers by the hepatitis B virus X protein.

We have used the chloramphenicol acetyltransferase (cat) gene expression system to study the effect of the X protein of hepatitis B virus (HBV) on viral enhancers. Plasmids containing the HBV enhancer and the core gene promoter linked to the cat gene were cotransfected with a plasmid containing the X gene into the human hepatoma cell line PLC/PRF/5. Our results indicate that the transfected X gene caused a trans-activation of the HBV enhancer. If a frameshift mutation or a deletion in the X structural gene was created, this trans-activation function was abolished. This result and the observation that the frameshift mutation did not alter the transcription of X mRNA suggest that the X protein is the trans-activating factor. Using similar techniques, we found that the X protein was also capable of trans-activating the simian virus 40 (SV40) and Rous sarcoma virus enhancers (pSV2cat and pRSVcat) in CV-1 cells. However, trans-activation of the SV40 enhancer by the X protein was not observed in COS-1 cells. By cotransfecting pSV2cat and the X gene with a plasmid containing either the intact SV40 genome, the SV40 genome devoid of the T-antigen (T-ag) gene, or only the T-ag gene, we demonstrated that SV40 T-ag can suppress trans-activation by the X protein. SV40 T-ag did not inhibit expression of the X gene or inactivate the X protein. The most probable mechanism of this inhibition is that T-ag competes with the X protein for a common target.

Degradation of starch and its hydrolytic products by oral bacteria.

Selected strains of oral bacteria were analyzed for their ability to degrade wheat starch, maltose, maltotriose, and maltoheptaose. S. sanguis IUOM-11M and JC804, S. mutans 6715, S. salivarius IUOM-8, A. viscosus IUOM-62, and A. naeslundii ATCC 12104 degraded all four substrates. S. mutans NCTC 10449 degraded starch, maltose, and maltotriose, while A. viscosus ATCC 15987 degraded starch and maltose, and S. sanguis SS34 degraded only maltose. L. casei IUOM-14 did not degrade any of the substrates. Analysis of starch degradation products from S. sanguis IUOM-11M and A. viscosus IUOM-62 demonstrated oligosaccharides, maltose, and trace amounts of glucose for the former and oligosaccharides, maltotriose, and maltose for the latter. S. sanguis IUOM-11M alpha-glucosidase (EC 3.2.1.20) demonstrated a pH optimum of 6.5 and greatly enhanced activity from maltose-cultured cells as compared with cells cultured in glucose or fructose. The presence of fructose in the growth medium prevented this enhancement of activity by maltose. Maltose inhibited sucrose-dependent synthesis of S. sanguis IUOM-11M insoluble polysaccharide and both primer-dependent and primer-independent synthesis of soluble polysaccharide. Maltoheptaose inhibited primer-dependent but not primer-independent soluble polysaccharide synthesis. Several oral bacteria have the ability to hydrolyze starch and to degrade further the products to acidogenic substrates. These products may also inhibit sucrose-dependent synthesis of polysaccharides, which enhances the production of the acidogenic substrate fructose. The results add further support to the growing body of evidence suggesting that caries-promoting properties of starch may be expressed only when starch is present in diets containing sucrose.