Non-Genetic Risk Factors of Alzheimer's Disease: An Updated Umbrella Review.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by intricate genetic and environmental etiology. The objective of this study was to identify robust non-genetic risk factors for AD through an updated umbrella review. METHODS: We conducted a comprehensive search of meta-analyses and systematic reviews on non-genetic risk factors associated with AD in PubMed, Cochrane, Embase, and Ovid Medline up to June 30, 2023. After collecting data, we estimated the summary effect size and their 95% confidence intervals. The degree of heterogeneity between studies was assessed using I2 statistics and a 95% prediction interval was determined. Additionally, we evaluated potential excess significant bias and small study effects within the selected candidate studies. RESULTS: The umbrella review encompassed a total of 53 eligible papers, which included 84 meta-analyses covering various factors such as lifestyle, diet, environmental exposures, comorbidity or infections, drugs, and biomarkers. Based on the evidence classification criteria employed in this study, two factors as convincing evidence (Class I), including rheumatoid arthritis (RA), potentially reduced the risk of AD, but diabetes significantly increased the risk of AD. Furthermore, three factors as highly suggestive evidence (Class II), namely depression, high homocysteine, and low folic acid level, potentially increased the risk of AD. CONCLUSION: Our findings highlight several risk factors associated with AD that warrant consideration as potential targets for intervention. However, it is crucial to prioritize the identified modifiable risk factors, namely rheumatoid arthritis, diabetes, depression, elevated homocysteine levels, and low folic acid levels to effectively address this complex neurodegenerative disorder.

[A comparative study of 3.0T (1)H-MRS for varying degrees of liver fibrosis in cynomolgus monkeys].

Objective: To investigate the changing rules with (1)H magnetic resonance spectroscopy ((1)H-MRS) in order to provide human research theoretical basis with varying degrees of liver fibrosis in cynomolgus monkeys. Methods: Liver fibrosis model in twenty-two cynomolgus monkey was successfully established with carbon tetrachloride (CCl(4)). Among them, fifteen cynomolgus monkey developed to early-stage liver cirrhosis (S4 stage). A comparative study was conducted in 15 cynomolgus monkeys that had fully developed liver fibrosis. The changing rules for varying degrees of liver fibrosis in cynomolgus monkeys were analyzed with (1)H-MRS. Supplementary methods: statistical analysis was performed using compatibility group design and analysis of variance for each research indicators. SNK-q test was used for pairwise comparison between the groups. The correlation between the 1H-MRS research indicators and the severity of liver fibrosis was analyzed by Spearman's rank correlation. Results: The Cho of (1)H-MRS was increased with the severity of liver fibrosis in cynomolgus monkeys. Moreover, there were statistically significant (P < 0.01) differences between liver fibrosis staging (S1 ~ S4) and normal liver tissue (S0 stage), severe liver fibrosis staging (S3 and S4) and mild to moderate liver fibrosis staging (S1 and S2). Compared with S0 stage, the peak value of lipid in S1 stage was significantly higher than that of S2 stage, and the peak value of lipid in S3 and S4 stage was significantly lower than that of S0 stage, and the differences between S1, S3, S4 and S0 stages were statistically significant (P < 0.01). The Cho/lipid ratio had gradually increased with the severity of liver fibrosis progression and the differences between groups were statistical significant (P < 0.01). Spearman's rank correlation coefficient between Cho / lipid ratio and pathological stage of liver fibrosis was 0.98 (P = 0.000). ROC curve analysis showed that Cho / lipid ratio was the most significant diagnostic indicator for liver fibrosis. The threshold values of CHO/lipid ratio were≥ 0.028, and≥ 0.131 (P < 0.01) for the diagnosis of liver fibrosis and early-stage cirrhosis. Conclusion: (1)H-MRS of the cynomolgus monkey liver fibrosis model changes rules regularly with the aggravation of severity of liver fibrosis. Among them, the Cho/lipid ratio is the most valuable indicator for the diagnosis of liver fibrosis staging, which may provide a theoretical basis for the study of human liver fibrosis.

[Clinical effect of one-stage total knee arthroplasty for knee osteoarthritis with femoral extra-articular deformity].

Objective: To investigate the application and efficacy of the one-stage total knee arthroplasty (TKA) of intra-articular compensation osteotomy in knee osteoarthritis(KOA) patients with extra-articular deformity (EAD). Methods: A retrospective study of 9 patients with end-stage KOA and EAD undergoing one-stage TKA from January 2014 to December 2017 in the First Affiliated Hospital of Zhejiang Chinese Medical University was performed. There were 3 males and 6 females with an average age of 56 years(range, 19-77 years);5 cases of simple coronal deformity (varus 10°-27°, mean 18.2°), 3 cases of sagittal deformity (recurvatum15°-35°, mean 22.6°), 1 case combined with coronal and sagittal deformity (varus 16°, recurvatum 31°); hemophilia dysplasia in 1 case, fracture malformation in 8 cases. Main outcome measures included the mechanical axis, range of motion (ROM) and Hospital for Special Surgery Knee Score (HSS). Results: The mean follow-up period was 33.2 months (range, 25-47 months). The mechanical axis angle was restored from 12.4°±4.1°to 1.4°±0.9°(t=7.954, P<0.01). The HSS was improved from 28±14 preoperatively to 87±7 postoperatively (t=-11.174, P=0.013). The ROM increased from 56°±22°to 99°±8° (t=-5.480, P=0.010). There was no complications such as joint instability, infection, fracture, common peroneal nerve injury and early prosthesis loosening. Conclusions: For KOA patients with femoral EAD, one-stage TKA with intra-articular compensatory osteotomy can effectively restore the mechanical axis and obtain satisfying joint function. Through a series of measures such as preoperative measurement, soft tissue evaluation and 3D printing, the accuracy of surgery can be improved and the difficulty of surgery can be reduced.

SPOP suppresses osteosarcoma invasion via PI3K/AKT/NF-κB signaling pathway.

OBJECTIVE: Speckle-type POZ protein (SPOP), is an E3 ubiquitin ligase adaptor that is frequently mutated in prostate and endometrial cancers. SPOP has been shown to be responsible for oncogene SRC-3 ubiquitination and proteolysis in prostate cancers.  However, whether SPOP plays a role in osteosarcoma (OS) is unknown. In this study, we investigated the inhibitory effect of SPOP on invasion and migration of OS cells. PATIENTS AND METHODS: Real-time PCR and Western blot were used to detect the expression of SPOP in human OS samples and cell lines. Short hairpin RNA (shRNA) was used to silencing the expression of SPOP. Small scale Real-time PCR screen was used to identify the matrix metalloproteases (MMP) family members responsible for the phenotype caused by SPOP depletion. Matrigel-coated invasion chambers were used to detect the invasion ability of SPOP in OS cells. RESULTS: We found that SPOP was down-regulated in clinic OS samples and cultured OS cells. Furthermore, we showed that silencing of SPOP promoted cell migratory and invasive ability of OS cells in vitro, whereas restored the expression of SPOP achieved the opposite effects. At the molecular level, we found that SPOP regulated the activity of "PI3K/Akt/NF-κB" signaling pathway in OS cells. CONCLUSIONS: Our results suggested that down-regulation of SPOP promoted OS cells migratory and invasive ability via modulating the "PI3K/Akt/NF-κB" signaling pathway. Thus, SPOP could be a promising drug target for the treatment of OS invasion.

A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host.

OBJECTIVES: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. MATERIALS AND METHODS: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. RESULTS: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). CONCLUSIONS: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

The imaging of neuroendocrine tumors using single photon emission computed tomography/computed tomography.

There have been several advances in technology over the past decade with the advent of hybrid imaging having a large impact on nuclear medicine, first with PET/CT and then more recently with SPECT/CT. Initial SPECT/CT systems used low dose but very low quality CT and except for attenuation correction offered no great advantage over reviewing SPECT and CT images side by side. More recently hybrid machines have become available and a series of studies have shown improved accuracy compared to SPECT alone with resulting changes in patient management. This has been true not only with somatostatin analogue imaging but also for demonstrating amine uptake using MIBG. Whilst PET/CT may be seen as the ideal, this may be less accessible due to the high cost and limited availability. In this case hybrid SPECT/CT offers hope for providing high quality and accurate imaging of neuroendocrine tumors.

Dosage prediction via estimation of shell thickness and concentration of drug carrier with microbubbles.

For drug delivery applications, dosage prediction before release and estimation after release are required functions. In this study, we attempted to establish a method to evaluate liposome concentrations and liposome shell thickness for dosage prediction. We use the Trilling model with parameter of phospholipids bilayers to simulate the frequency responses under the different acoustic pressure and establish an experimental protocol to evaluate the liposome concentrations and the liposome shell thickness. Our results illustrate the changes on the signal strength for different concentrations and show that it is relatively stable to estimate the concentrations when the cycles are lower (15 cycles). Besides, it is verified that the second harmonic signal is more sensitive in analyzing different concentrations. On the other hand, it is proved that the liposome shell thickness affect signal strength and thinner thickness will increase the second harmonic response. Therefore, in accordance with the theoretical and experimental results, we would be able to estimate the concentration and the shell thickness of the liposomes. By numerical analysis methods, dosage prediction would be built.

Connexin 36 is expressed and associated with zonula occludens-1 protein in PC-12 cells.

Connexin 36 (Cx36) is the predominant connexin isoform expressed in the mammalian neurons of the central nervous system (CNS). PC-12 cells, a neuronal-like cell line, are widely used for neuron functional studies. Many connexins have been shown to interact with zonula occludens-1 protein (ZO-1), a tight junction associated with protein. The present study is intended to investigate whether Cx36 is expressed in PC-12 cells and is associated with ZO-1. Cx36 transcripts were amplified and verified by RT-PCR. 2.9 kb Cx36 mRNA was detected in PC-12 cells through Northern blot hybridization. Western blotting showed a 36-kDa protein band in the homogenates of PC-12 cells. Immunofluorescence labeling revealed that Cx36 was present in cell-cell contacts of PC-12 cells and colocalized with ZO-1. The association of Cx36 and ZO-1 in PC-12 cells was also demonstrated by coimmunoprecipitation. In conclusion, PC-12 cells express Cx36 mRNA and Cx36 proteins that are associated with ZO-1. These results enhanced our understanding of the function of Cx36 in PC-12 cells.

Reevaluation of the role of early trypsin activity in the transcriptional activation of the late trypsin gene in the mosquito Aedes aegypti.

In the female mosquito Aedes aegypti, trypsin expression is largely biphasic. Early trypsin synthesis, which is regulated at the translational level relative to feeding, peaks in the first few hours post-blood meal. Late trypsin expression is regulated at the transcriptional level, and peaks 18-24h post-blood meal. It was proposed that early trypsin activity released unknown factors during digestion of a meal that caused activation of transcription of the late trypsin gene. This connection between early trypsin activity and late trypsin expression was dependent on the fact that feeding a single trypsin inhibitor, soybean trypsin inhibitor (STI), which blocked early trypsin activity, also blocked late trypsin expression. We show in this study that feeding different trypsin inhibitors which effectively blocked early trypsin activity did not result in reduced late trypsin expression. We also found that a different lot of STI failed to cause inhibition of late trypsin transcription, although it was effective in inhibiting early trypsin activity. In addition, using RNAi methodology to reduce the level of early trypsin expression had no effect on the level of late trypsin expression. We conclude that early trypsin activity is not necessary for the transcriptional activation of late trypsin and that the previous results were due to the effect of a cytotoxic agent present in some, but not all preparations of STI.

Hematopoietic differentiation of rhesus monkey embryonic stem cells.

Several lines of embryonic stem cells (ESC) have been established from rhesus monkey blastocysts. We have examined two of these cell lines for their potential for generating hematopoietic progenitors in cell culture, and we identified culture conditions, including supplementation with bone morphogenetic proteins (BMP), that result in hematopoietic differentiation of rhesus ESC with high efficiency. We have also characterized the resulting hematopoietic progenitor cells for their patterns of gene expression, as compared to those of hematopoietic progenitor cells harvested from rhesus monkey bone marrow. Of more than 60 genes examined in this manner, CD34+/CD38- cells derived from embryonic stem cells and those obtained from bone marrow demonstrated very similar patterns of gene expression. However, with integrin alphaL, IL-6 receptor, and flt-3 gene expression was greatly diminished or absent in CD34+/CD38- cells derived from the ESC, whereas the bone marrow-derived progenitors showed substantial expression of all of these genes. When the same type of comparison was done with mouse (D3 and CCE) as well as human (H1) embryonic stem cells, in each case comparing ESC-derived hematopoietic progenitors with those harvested from bone marrow, the only consistent deficiency of gene expression was that of flt-3. In hematopoietic precursors derived from mouse ESC, globin-gene expression has previously been shown to be a useful index of the embryological maturity of the cells, and we also examined globin-gene expression in rhesus monkey ESC-derived hematopoietic precursor cells, using a semiquantitative technique. CD34+/CD38- cells demonstrated expression of the epsilon- and gamma-globin genes, but negligible levels of beta globin, suggesting that these cells were at the developmental stage in which the yolk sac and fetal liver are the primary sites of hematopoiesis.

Contiguous arrangement of p45 NFE2, HnRNP A1, and HP1 alpha on mouse chromosome 15 and human chromosome 12: evidence for suppression of these genes due to retroviral integration within the Fli-2 locus.

Fli-2 is a common site of proviral integration in multistage erythroleukemia cells induced by Friend murine leukemia virus (F-MuLV) or the polycythemia strain of Friend leukemia virus (FV-P). Previously, we reported that integration of Friend virus into the Fli-2 locus in CB3, an erythroleukemia cell line that harbors a homozygous inactivation of the Fli-2 locus, results in the loss of expression of two genes encoding the 45-kDa subunit of the erythroid-specific nuclear factor p45 NFE2 and the splicing factor HnRNP A1. Here, we report the identification of a third gene, Heterochromatin protein 1 (HP1alpha, also known as CBX5), which is located downstream of HnRNP A1, and p45 NFE2. Northern blot analysis revealed that the expression of HP1alpha, along with p45 NFE2 and HnRNP A1, is either undetectable or substantially reduced in CB3 cells, suggesting that HP1alpha expression is also regulated by proviral insertion within the Fli-2 locus in CB3 cells. Because p45 NFE2 was previously mapped to mouse chromosome 15, our results demonstrate that HP1alpha and HnRNP A1 are also located on mouse chromosome 15 and that the p45 NFE2, HnRNP A1, and HP1alpha genes are arranged contiguously. Contiguous arrangement of these three genes was also detected in man; this consequently localizes HP1alpha to human chromosome band 12q13.

Lineage-specific mechanism of drug and radiation resistance in melanoma mediated by tyrosinase-related protein 2.

A major obstacle in the clinical management of malignant melanoma is its intrinsic resistance to chemotherapy and radiation therapy. Consequently, most patients with melanoma often do not respond to conventional anticancer therapy in a clinically significant manner. Recent advances in cancer research have provided new insights into the mechanisms of intrinsic resistance in melanomas. We have recently reported that the over-expression of tyrosinase-related protein 2 (TYRP2), an enzyme that is well characterized for its function in melanin synthesis, is associated specifically with resistance to DNA damaging drugs and radiation treatment. This review will summarize our findings as well as discuss the possible mechanisms by which TYRP2 over-expression contributes to intrinsic resistance in human malignant melanoma.

Tyrosinase-related protein 2 as a mediator of melanoma specific resistance to cis-diamminedichloroplatinum(II): therapeutic implications.

A major obstacle in the systemic treatment of advanced malignant melanoma is its intrinsic resistance to conventionally used chemotherapeutic agents. In order to investigate the mechanisms of this intrinsic resistance, we have previously utilized retroviral insertional mutagenesis on an early-stage, drug sensitive human melanoma cell line (WM35) to establish mutated cell lines that exhibited increased resistance to cis-diammi-nedichloroplatinum(II) (CDDP). Here, we demonstrate that this increased resistance to CDDP is mediated by the over-expression of tyrosinase-related protein-2 (TYRP2), an enzyme that normally functions in the biosynthesis of the pigment, melanin. Northern and Western blot analyses revealed that the expression of TYRP2 in the virally-derived cell lines as well as in a panel of human melanoma cell lines positively correlated with their levels of resistance to CDDP. Furthermore, enforced expression of TYRP2 in WM35 cells by transfection elevated their resistance to CDDP. The increased CDDP resistance in the virally-derived clones and TYRP2 transfectants was accompanied by a reduction in CDDP-induced apoptosis. Interestingly, the virally-derived CDDP-resistant clones also showed cross resistance to carboplatin and methotrexate, but not taxol, suggesting that TYRP2 over-expression may confer resistance specifically to DNA damaging agents. Overall, these results demonstrate a novel mechanism of drug resistance in human melanoma cells that is mediated by the over-expression of TYRP2. Since TYRP2 is expressed only in cells of melanocytic lineage, this may represent the first report of a lineage-specific mechanism of drug resistance. In summary, these findings suggest a significant role for TYRP2 in the intrinsic drug resistance phenotype of human melanoma cells and may have important implications in the development of chemosensitization strategies for the clinical management of this disease.

[Timing changes of apoptosis and proliferating cells nuclear antigen after intra-arteiral infusion chemotherapy for nasopharyngeal carcinoma].

OBJECTIVE: To investigate the timing changes of apoptosis (APO) and PCNA after intra-arterial infusion chemotherapy for nasopharyngeal carcinoma. METHOD: Ten patients were subjected to percutaneous superficial temporal artery catheterization and infusion of anticancer drugs: 5-Fu, DDp, BLM, nicotinamide. The biopsy of nasopharyngeal tumor tissues was performed before chemotherapy and 7 days after chemotherapy. Apoptotic cells were examined by TOT-mediated DUTP-fluorescein and labeling. The expression of proliferating cells nuclear antigen (PCNA) was detected with immunohistochemical staining. RESULT: AI of nasopharyngeal cancer cells before chemotherapy and 7 days after chemotherapy was 0.52% and 1.42%, PI was 42.4% and 51.2%. CONCLUSION: Intra-arterial infusion chemotherapy not only induced apoptosis effectively, but also inhibited temporarily tumor cells proliferation in patients. The curative surgical treatment should be performed as soon as possible after chemotherapy.

Elevated basal and thapsigargin-stimulated intracellular calcium of platelets and lymphocytes from bipolar affective disorder patients measured by a fluorometric microassay.

BACKGROUND: A number of investigators have reported finding elevated basal and stimulated intracellular calcium levels in the platelets or lymphocytes of bipolar disorder patients. METHODS: Intracellular calcium was measured by a micro fura-2 fluorometric method in the platelets and lymphocytes of 30 affective disorder patients and 14 control subjects. RESULTS: We observed significantly elevated basal calcium concentrations in bipolar patient platelets and lymphocytes compared to control subjects. Bipolar patient platelet calcium responses to thrombin, serotonin, and thapsigargin were also significantly greater than control subjects. The peak calcium levels of lymphocytes of bipolar patients were greater than control subjects only when stimulated by thapsigargin. There were significant differences between bipolar and unipolar patients in basal and thapsigargin-stimulated calcium measures but not between bipolar I and bipolar II patients. Unmedicated versus medicated calcium measures were not significantly different. We also found little correlation between calcium measures and the severity of mood rating. CONCLUSIONS: Using this method, we were able to confirm and extend the work of others, indicating altered intracellular calcium homeostasis in the blood cells of bipolar disorder patients. In addition, our data suggest that storage operated calcium channels may be the source of the elevated intracellular calcium in platelets and lymphocytes of bipolar patients.

Juvenile myelomonocytic leukemia (JMML) with the hematologic phenotype of severe beta thalassemia.

A 3-year-old Filipino-American child with recurrent fever, splenomegaly, anemia, and thrombocytopenia, was found to have a hemoglobin F level of 76.9%. His reticulocyte count was elevated (4.3%), and erythroblasts were present in his peripheral blood. The child's erythrocytes were microcytic (MCV 66.9 fl) but his serum ferritin level was normal. His bone marrow at initial presentation demonstrated normal cellularity without an increase in blast cells. The disease progressed with worsening anemia, leukocytosis, and thrombocytopenia, with increased blasts in his marrow and the appearance of a mediastinal mass. His liver, spleen, and lymph nodes were found to be infiltrated with myeloblasts, supporting a diagnosis of juvenile myelomonocytic leukemia (JMML). Analysis of the child's Hb F showed a Ggamma/Agamma ratio of 2.2, which was within the characteristic range for JMML. A globin synthesis study using blood reticulocytes showed an alpha/non-alpha globin synthesis ratio of 2.24, typical of severe homozygous beta thalassemia. Southern blot analysis of blood-leukocyte DNA from the patient and his parents demonstrated no apparent abnormality in the beta-globin gene promoter or coding regions. The elevated level of Hb F in this child with JMML appeared to be part of an acquired Cooley's anemia-like hematologic phenotype.

A naturally occurring amino acid substitution of the human serotonin 5-HT2A receptor influences amplitude and timing of intracellular calcium mobilization.

Recently, two naturally occurring amino acid substitutions were identified in the C-terminal region of the serotonin 5-HT2A receptor. One of these, His452Tyr, has a rarer allele Tyr frequency of 9%. If 452Tyr alters 5-HT2A function, it would thus be a candidate allele for human neurobehavioral variation. The present study was designed to evaluate the potential influence of the 452His and 452Tyr alleles on cellular 5-HT2A functions. Platelet 5-HT2A binding and 5-HT-induced Ca2+ response were compared in eight 452His/452His homozygous and eight 452His/452Tyr heterozygous individuals matched for sex, age, and diagnosis (all were patients with seasonal affective disorder). There was no difference in 5-HT2A binding measured using 125I-lysergic acid diethylamide. Nor were levels of G-protein subunits or PKC alpha, delta, epsilon, or zeta significantly altered. However, when Ca2+ response was stimulated by 2, 5, 10, or 25 microM 5-HT, significant differences were found. In 452His/452Tyr heterozygotes, 452Tyr was associated with both smaller peak amplitude in Ca2+ mobilization and a different time course of response, with slower peak latency and longer half-time in 452His/452Tyr heterozygotes compared with 452His/452His homozygotes. The overall difference in the response of the 5-HT2A receptor in individuals with 452Tyr was a blunting of the shape of the Ca2+ mobilization peak. The data reported here suggest that the primary sequence of this intracellular domain is important in function of the receptor and that the 452His and 452Tyr 5-HT2A alleles should be carefully evaluated for effects on human neurobehavioral variation.

Effect of dietary fat or tamoxifen on the expansion of cells harboring Ha-ras oncogenes in mammary glands from methylnitrosourea-treated rats.

Diets containing high levels of fat enhance the formation of methylnitrosourea (MNU)-induced mammary gland adenocarcinomas in rats, while administration of the antiestrogen tamoxifen decreases the incidence of these tumors. It is not known, however, at what stage during tumor development the fat or tamoxifen exert their effects. Here we have used a PCR/liquid hybridization and gel retardation assay to determine the effects of dietary fat and tamoxifen on the growth rate of cells harboring an Ha-ras oncogene in the mammary glands of rats at various times following MNU administration. Glands from animals on a high-fat diet had significantly higher mutant cell fractions than those on a low-fat diet at both 30 and 75 days following MNU treatment. In contrast, there was no difference between the mutant cell fractions of tamoxifen-treated animals and controls at either 30 or 70 days. These results suggest that dietary fat promotes tumor formation early in carcinogenesis by stimulating the growth of cells harboring Ha-ras mutations, while tamoxifen delays the appearance of tumors either by acting as a tumoristatic or tumoricidal agent, or by acting to eliminate or retard the growth of preneoplastic cells just prior to the emergence of tumors.