Alterations in cell cycle regulation in mouse skin tumors

Using a modified tip of the atomic force microscope (AFM), we harvested several strands of genomic DNA from a nanometer region of mouse chromosomes. We have also co-developed a random PCR method to amplify the recovered genomic DNA, in which a single DNA molecule of several kilobasepairs could be amplified efficiently. A subsequent fluorescence in situ hybridization (FISH) indicated that the amplified DNA originally came from the tip-manipulated regions of mouse chromosomes. Several fragments containing unique sequences were identified using Southern hybridization after subcloning the PCR products into pUC18 plasmid. The present results showed a potential application of AFM to genomic analysis.     

The role of receptors in multistage carcinogenesis

The principal characteristic of neoplasia is its inherited alteration of genetic expression. The regulation of gene expression may be altered both by mutational events and by environmental mediators. During carcinogenesis the permanent alterations in genetic expression resulting from mutations occur primarily during the final stage of progression when biological malignancy becomes evident. During the preceding reversible stage of promotion, alteration and genetic expression are the result of the chronic stimulation of an altered (initiated) cell responding to the environmental mediator or promoting agent. A major mechanism of this effect occurs by receptors exhibiting specificity for the mediator and for their interaction with the genome. Withdrawal of the promoting agent prior to the genetic alterations characteristic of the stage of progression leads to a reversal of the effects of the promoting agent and the death by apoptosis of most cells in the stage of promotion. Carcinogenesis mediated by the chronic ligand (promoting agent)-receptor interaction increases the probability of the development of the stage of progression; thus alteration or prevention of the stage of promotion by removal of the promoting agent or inhibition of its action remains the best opportunity for cancer prevention. Application of the reversible promoting agent-receptor interaction to specific environmental circumstances where such plays a major role can lead to a more rational risk estimation of promoting agents for the human population.     

Chronic ultraviolet exposure-induced p53 gene alterations in Sencar mouse skin carcinogenesis model

Alterations of the tumor suppresser gene p53 have been found in ultraviolet radiation (UVR) related human skin cancers and in UVR-induced murine skin tumors. However, links between p53 gene alterations and the stages of carcinogenesis induced by UVR have not been clearly defined. We established a chronic UVR exposure-induced Sencar mouse skin carcinogenesis model to determine the frequency of p53 gene alterations in different stages of carcinogenesis, including UV-exposed skin, papillomas, squamous-cell carcinomas (SCCs), and malignant spindle-cell tumors (SCTs). A high incidence of SCCs and SCTs were found in this model. Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. DNA was isolated from 40 paraffin-embedded normal skin, UV-exposed skin, and tumor sections. The p53 gene (exons 5 and 6) was amplified from the sections by using nested polymerase chain reaction (PCR). Subsequent single-strand conformation polymorphism (SSCP) assay and sequencing analysis revealed one point mutation in exon 6 (coden 193, C-->A transition) from a UV-exposed skin sample, and seven point mutations in exon 5 (codens 146, 158, 150, 165, and 161, three C-->T, two C-->A, one C-->G, and one A-->T transition, respectively) from four SCTs, two SCCs and one UV-exposed skin sample. These experimental results demonstrate that alterations in the p53 gene are frequent events in chronic UV exposure-induced SCCs and later stage SCTs in Sencar mouse skin.     

Ultraviolet radiation and skin carcinogenesis

A case report of boutonneuse fever with pulmonary complications in a patient with non-Hodgkin's lymphoma (NHL) is described. The patient was hospitalized for persistent hypertermia and marked dyspnea, with radiographic findings of bilateral involvement of the lungs. The confirmation of the diagnosis was obtained by means of serum analyses (Weil-Felix serodiagnosis and IFA); the patient responded to doxycycline with progressive improvement of her general health condition. In this case the occurrence of a NHL could justify the lower reactivity and the facilitated diffusion of rickettsiosis in the patient.     

p53 expression in skin carcinogenesis and its relationship to cell proliferation and tumour growth

The immunoreactivity of p53 protein was studied in relation to tumour development, histopathological characteristics, cell proliferation, and basement membrane organisation following the induction of skin carcinogenesis in tumour-sensitive and -resistant mouse strains by ultraviolet (UV) irradiation or 7,12-dimethylbenz(a)anthracene (DMBA). In non-neoplastic skin exposed to UV irradiation or DMBA, p53 immunoreactivity was observed in nearly 50% of the basal layer cells. These cells were morphologically and histochemically indistinguishable from the p53-negative cells, occurring similarly in the tumour-producing and the tumour-negative mouse strains and regardless of subsequent tumour formation. In induced epidermal hyperplasia and in benign tumours, p53-positive and proliferating cells constituted 40-50% of all cells in the basal layer, while superficial cells were p53 negative. In dysplastic epidermis, p53-positive cells and proliferating cells were seen in all cell layers. In the case of squamous cell carcinomas, p53-positive proliferating cells in differentiated neoplasms were localised close to the basement membrane and, more frequently, in border areas showing invasion and basement membrane destruction. In horn cysts, centrally located cells were non-proliferating and p53 negative. In moderately differentiated neoplasms, proliferating cells were located closer to the basement membrane, while p53-positive cells were distributed diffusely in the neoplasm. In poorly differentiated neoplasms, p53-positive cells were more common than proliferating cells and were arranged in a diffuse pattern. The results showed that the number and location of p53-positive cells depended upon histology, with a close relationship to tumour type and degree of malignancy, but not on the mode of induction, nor on the animal strain or the relationship to subsequent tumour formation.     

Morphofunctional changes in animal skin during combined administration of zinc sulfate and vitamin A

Morphological and histochemical skin properties were studied in male rats in single and successive peroral injections of Zinc sulphate and vitamin A. Long term (25 d) peroral administration of vitamin A 2 mg/100 g in dose was shown to cause hypozincaemia. Zinc sulphate and vitamin A are capable of influencing proliferation and protein metabolism of epidermal cells and number of lymphocytes and mast cells in dermis. With the aim of the drugs named thickness of epidermis, ratio of its layers thickness as well as the rate of protein synthesis in germinal layer of epidermis can be changed purposefully. These parameters depend both on the drug injection technique (single, separated or successive injections) and time periods. Perspective of obtaining a complexed biocoordinative compound on the base of vitamin A and microtrace element of Zinc is discussed.     

Regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation

Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.     

Expression of heat shock proteins in mouse skin during wound healing

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.     

Temporal variation in cellular proliferation during recornification of mouse tail skin

The influence of the time of injury on subsequent epidermal regeneration is unknown. Epidermal cell proliferation of tail skin in C57BL/6J mice in response to tape stripping was followed for 7 days by radiolabelled thymidine incorporation and autoradiography. The homeostatic labelling index (LI) of the basal epidermis of unmanipulated, unwounded (control) animals was 7.6% and did not vary depending on the time of day. Tape stripping increased the LI of epidermal basal cells 110% above control values 24 h after injury. Labelling indexes of epidermal basal cells in the skin adjacent to the wounded area were 7.0%. Basal cell DNA synthesis stimulated by wounding exhibited a distinct temporal variation at 24 h postinjury, with tail skin wounded at 12.00 h found to be 275% greater than control values and elevated 78% from LIs recorded at any other time point. This temporal spike was due to the time of day at which wounding occurred rather than the time point when the LI was determined. Mice wounded at 12.00 h and terminated 27 h later (15.00 h) had LIs that were 52% greater than wounds created at 09.00 h and examined at 12.00 h the following day. Higher levels of DNA synthesis in tail skin injured at 12.00 h compared to wounding at 09.00 h was detected 12-48 h after injury. Furthermore, DNA synthesis in wounds created at 12.00 h returned to baseline levels 1-2 days earlier than tail skin wounded at 09.00 h. Investigation of other strains of mice detected differences in radiolabelling of epidermal basal cells 24 h after tape stripping at 12.00 h or 09.00 h in CD-1 and BALB/cJ mice, but not in the C3H/HeJ strain. These results indicate: (a) there is no diurnal variation in the LI of mouse tail skin under normal homeostatic conditions (b) tape stripping is a potent stimulator of basal cell turnover in the epidermis (c) the time of wounding determines the magnitude of the increase in the LI of basal cells following injury, and (d) the proliferative response to wounding of the tail is dependent on the strain of mouse.     

Continuous systemic secretion of a lysosomal enzyme by genetically modified mouse skin fibroblasts

Cholera toxin from Vibrio cholerae has been shown to increase the secretory immune response when given orally with some antigens in mice and rabbits. The present study was designed to determine if cholera toxin was also an effective mucosal adjuvant in chickens. Tetanus toxoid was chosen as a model antigen, and response was measured by enzyme-linked immunosorbent assays of intestinal excreta, bile samples, and serum samples. Chickens given 20 micrograms of tetanus toxoid had a significant suppression of response when 20 or 50 micrograms of cholera toxin was given. Chickens given 100 micrograms of tetanus toxoid appeared to be mucosally tolerant, but there was apparent abrogation of the mucosal tolerance when 20 or 50 micrograms of cholera toxin was given. Cholera toxin apparently failed as a mucosal adjuvant in chickens but may have some anti-mucosal tolerogenic effect.     

Alterations in the enzyme activity and protein contents of protein disulfide isomerase in rat tissues during fasting and refeeding

Elevated level of cellular lipid peroxidation can increase the incidence of vascular disease. The mechanism by which ketosis causes accelerated cellular damage and vascular disease in diabetes is not known. This study was undertaken to test the hypothesis that elevated levels of ketone bodies increase lipid peroxidation in endothelial cells. Human umbilical venous endothelial cells (HUVEC) were cultured for 24 h at 37 degrees C with ketone bodies (acetoacetate, beta-hydroxybutyrate). Acetoacetate, but not beta-hydroxybutyrate, caused an increase in lipid peroxidation and growth inhibition in cultured HUVEC. To determine whether ketone bodies generate oxygen radicals, studies using cell-free buffered solution were performed. They showed a significant superoxide dismutase (SOD) inhibitable reduction of cytochrome C by acetoacetate, but not by beta-hydroxybutyrate, suggesting the generation of superoxide anion radicals by acetoacetate. Additional studies show that Fe2+ potentiates oxygen radical generation by acetoacetate. Thus, elevated levels of ketone body acetoacetate can generate oxygen radicals and cause lipid peroxidation in endothelial cells, providing a possible mechanism for the increased incidence of vascular disease in diabetes.     

Transforming growth factor-beta expression in mouse lung carcinogenesis

Transforming growth factor-beta (TGF-beta) is a multifunctional growth modulator that inhibits the proliferation of many epithelial cells while stimulating the proliferation of most fibroblasts. To examine the role of TGF-beta in mouse lung chemically induced tumorigenesis, expression of the TGF-beta 1, -beta 2, and -beta 3 proteins was examined in A/J mice treated with the carcinogen urethane to induce lung adenomas using immunohistochemical staining analysis. Immunostaining for the TGF-beta ligands was detected in the epithelium of the bronchioles of untreated A/J mice with immunostaining being more intense for TGF-beta 1 than for TGF-beta 2 and TGF-beta 3; immunostaining for each TGF-beta ligand was also detected in the bronchiolar epithelium of urethane-treated A/J mice at levels similar to untreated mice. Immunostaining for the TGF-beta ligands was also detected in adenomas by 2 months; staining for TGF-beta 1, -beta 2, and -beta 3 in adenomas was detected at levels comparable with bronchioles. Following treatment with urethane for 8 months, immunostaining for TGF-beta s 1, 2, and 3 in bronchioles persisted at levels comparable to that in normal bronchioles and also persisted in adenomas, with staining for the TGF-beta ligands being very prominent on the edge of the tumor. Expression of TGF-beta 1 mRNA was examined in urethane-treated mouse lung tissue using Northern blot hybridization; here, expression of TGF-beta 1 mRNA increased 2-fold in 3-month urethane-treated lung tissue and an additional 2.5-fold by 8 months following urethane administration. Expression of TGF-beta 1 mRNA was also examined in nontumorigenic and tumorigenic mouse lung cells; in these cells, expression of TGF-beta 1 mRNA was higher in the tumorigenic cells than in the nontumorigenic cell line. These data show that there is an increase in expression of TGF-beta 1 during tumorigenesis and suggest that TGF-beta may play an important role in mouse lung carcinogenesis induced by urethane.     

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.     

p96, a MAPK-related protein, is consistently downregulated during mouse mammary carcinogenesis

Differential display PCR [DD-PCR] was applied to identify mRNAs differentially expressed between two consecutive stages of an in vivo model of mouse mammary carcinogenesis. The extended life 12 [EL12] and transformed mammary 12 [TM12] outgrowths differ in morphology, ovarian hormone dependence, and tumorigenicity, yet the TM12 outgrowth arose spontaneously from the EL12 outgrowth. A fragment of the mouse p96 gene was identified using DD-PCR. The differential expression of p96 was confirmed using RNase protection assays. Examination of the RNA expression patterns of the p96 isoforms during normal mammary gland development showed high levels in the involuting mammary gland and in preneoplastic hyperplasias. In contrast, p96 isoform mRNA levels were consistently decreased in mammary tumors derived from the in vivo hyperplasias. Examination of p96 protein levels revealed a decrease in p96 protein in a number of mammary tumors as compared to their hyperplastic precursors further supporting the observations that p96 gene expression is consistently downregulated in mammary tumors. The functional activity of p96 protein has not been resolved, however the observation that p96 gene expression is downregulated in two different tumor systems (human ovarian tumors and mouse mammary tumors) warrants more extensive investigation on its role in normal and neoplastic cell growth.     

Alterations in eighteen-carbon saturated, monounsaturated and polyunsaturated fatty acid peroxisomal oxidation in mouse brain during development and aging

PURPOSE/OBJECTIVES: To provide an update on the breast cancer genes BRCA1 and BRCA2 and to review available primary preventive options. DATA SOURCES: Published articles, abstracts, and clinical experience. DATA SYNTHESIS: While genetic testing will help identify a cadre of women at high risk for breast cancer development, it also will raise many psychosocial and ethical issues, including if and when to be tested and what patients and healthcare professional should do with the information. CONCLUSIONS: The only currently available putative form of primary prevention is prophylactic mastectomy. Diet and the use of tamoxifen remain areas for future research. IMPLICATIONS FOR NURSING PRACTICE: Nurses can play an important role in educating patients who face difficult decisions surrounding genetic testing and primary prevention modalities. Nurses also can design and conduct much needed research in these areas.     

Developmental patterns of acid hydrolases during differentiation of fetal mouse skin

A dye reduction method was developed for estimating total aerobic and/or psychrotrophic bacterial counts in ground beef. The method is based on color changes in indicator disks placed on the meat surface.     

Inhibitory effect of cycloartenol ferulate, a component of rice bran, on tumor promotion in two-stage carcinogenesis in mouse skin

Sentinel lymph node (SN) biopsy in primary breast cancer seems to be an alternative to complete axillary lymph node dissection for evaluation of the axillary lymph node status in selected patients. Following evaluation of the technique of SN biopsy, we applied SN biopsy clinically to 23 selected patients. A lymphoscintigraphy was performed preoperatively to evaluate lymphatic drainage. The SN was identified in the operating room by use of a hand-held gamma camera and a blue vital dye. The SN was found in all 23 patients. In 17/23 the SN was tumor free and no axillary dissection was performed; 6/23 showed a positive SN and complete axillary dissection was done. Intraoperative lymphatic mapping and SN biopsy may be the treatment of choice for evaluation of axillary node status in selected patients, for it combines accurate nodal staging and low morbidity.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal outbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B-lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., 3H-thymidine incorporation) and cell membrane level of interleukin-2 receptors (IL-2R; determined by flow cytometry) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

Alterations in reverse cholesterol transport associated with programmable implantable intraperitoneal insulin delivery

OBJECTIVE: As the population ages, the care of older persons becomes more important. At the same time, practice guidelines that provide recommendations for appropriate care are being published in greater numbers. The purpose of this work is to determine the proportion of guidelines that contain specific information about older persons. DESIGN: Through a random sample of published guidelines listed in the AMA Directory of Practice Parameters, 1992 Edition, we determined the proportion of guidelines that contain specific age-related information. We also determined if, over time, there was a difference in the proportion of practice guidelines containing information about older persons. RESULTS: 45.9% (95% CI, range 33.4-58.4) of guidelines that could conceivably pertain to older persons contain no age information; 24.6% (95% CI, range 13.8-35.4) of guidelines contain information only about persons less than 65 years of age; 29.5% (95% CI, range 18.1-41.0) of guidelines contain specific information about older persons. Moreover, there were no secular trends in the proportion of guidelines pertaining to older persons. CONCLUSIONS: Only a minority of practice guidelines contain information about older persons. Possible causes and solutions to this shortfall are discussed.