Abstract: INTRODUCTION: Breast cancer patients with HER2/neu overexpression have poor outcomes with a decrease in disease-free survival (DFS) and overall survival. The biology of HER2/neu overexpression in breast tumors in African-American and Latina women is poorly understood. The purpose of this study is to understand the clinical significance of activated Akt (phospho-Akt or pAkt) expression in breast tumors from African-American and Latina patients with corresponding tissue HER2/neu overexpression. Cellular and molecular studies have shown that activation of the cell signaling phosphatidylinositol-3-kinase/Akt cascade via the HER2/neu and other receptor tyrosine kinases induces cell proliferation. METHODS: A total of 234 African-American and Latina patients were selected retrospectively. From this group, 141 tumor tissue samples were analyzed for tissue pAkt by immunohistochemistry (IHC). This cohort consisted of 46 HER2/neu-positive (3+ by IHC) and 95 HER2/neu-negative tumors. The prognostic value of activated tissue Akt in relation to HER2/neu overexpression for DFS was determined. RESULTS: Patients with low pAkt and HER2-negative tumors had the best DFS. As expected, HER2/neu-overexpressing tumors with low pAkt had a decrease in DFS. Similarly, those with high pAkt and HER2-negative tumors also had poor DFS. However, those with an increase in both HER2 and pAkt had the worst DFS. An increase in pAkt was significantly associated with HER2/neu-positive and lymph node-positive breast tumors. Tumors with high HER2 and high pAkt were metastatic. Multivariate analysis demonstrated that, in addition to the common risk factors such as larger tumor size, lymph node involvement, estrogen receptor/progesterone receptor-negative tumors, and HER2/neu-positive tumors, overexpression of pAkt significantly was associated with a decrease in 5-year DFS. A decrease in DFS with an increase in pAkt was observed in both HER2/neu-positive and -negative groups. However, the DFS was similar between HER2/neu-positive/pAkt-negative and HER2/neu-negative/pAkt-positive groups. CONCLUSION: Our data suggest that there may be differences in tumor phenotypes within the HER2/neu-overexpressing breast cancer patients. The overexpression of pAkt may be a powerful prognostic marker for predicting DFS and overall survival of breast cancer patients.
Abstract: A major outcome from Taxol treatment is induction of tumor cell apoptosis. However, metabolic responses to Taxol-induced apoptosis are poorly understood. In this study, we hypothesize that alterations in specific amino acid transporters may affect the Taxol-induced apoptosis in breast cancer cells. In this case, the activity of the given transporter may serve as a biomarker that could provide a biological assessment of response to drug treatment. We have examined the mechanisms responsible for Taxol-induced neutral amino acid uptake by breast cancer cells, such as MCF-7, BT474, MDAMB231 and T47D. The biochemical and molecular studies include: (1) growth-inhibition (MTT); (2) transport kinetics: (3) substrate-specific inhibition; (4) effect of thiol-modifying agents NEM and NPM; (5) gene expression of amino acid transporters; and (6) apoptotic assays. Our data show that Taxol treatment of MCF-7 cells induced a transient increase in Na(+)-dependent transport of the neutral amino acid transporter B0 at both gene and protein level. This increase was attenuated by blocking the transporter in the presence of high concentrations of the substrate amino acid. Other neutral amino acid transporters such as ATA2 (System A) and ASC were not altered. Amino acid starvation resulted in the expected up-regulation of System A (ATA2) gene, but not for B0 and ASC. B0 was significantly down regulated. Taxol treatment had no significant effect on the uptake of arginine and glutamate as measured by System y(+) and X(-) (GC) respectively. Tunel assays and FACS cell cycle analysis demonstrated that both Taxol- and doxorubicin-induced upregulation of B0 transporter gene with accompanying increase in cell apoptosis, could be reversed partially by blocking the B0 transporter with high concentration of alanine, and/or by inhibiting the caspase pathway. Both Taxol and doxorubicin treatment caused a significant decrease in S-phase of the cell cycle. However, Taxol-induced an increase primarily in the G2 fraction while doxorubicin caused increase in G1/G0 together with a small increase in G2. In summary, our study showed that Taxol induced apoptosis in several breast cancer cells results in activation of amino acid transporter System B0 at both gene and protein level. Similar response was observed with another chemotherapeutic agent Doxorubicin, suggesting that this increase is in response to apoptosis, and not only due to changes in cell cycle related events.
Abstract: We sought to assess the significance of an incidental finding of colorectal wall thickening (CRWT) on computed tomography (CT) scan in African-American and Hispanic patients. We retrospectively reviewed charts of African-American and Hispanic patients from January 1994 to December 2005. Those patients were included in whom the colonoscopy was performed due to incidental CRWT on CT scan. Patients with a history or a family history of colorectal malignancy, inflammatory bowel disease, or colorectal surgery, with an incomplete colonoscopic examination, or <18 years of age were excluded. Endoscopic and pathological findings were abstracted. Thirty-two patients met the criteria. Endoscopic examination was abnormal in 21 (65.6%). The positive predictive value of CRWT for abnormal endoscopic examination was 65.6%. Abnormal endoscopic examination revealed diverticulosis in 9 (43%), erythematous mucosa in 8 (38%), polyps in 6 (29%), mass in 2 (9%), thickened folds in 1 (5%), and diverticulitis in 1 (5%). Histopathological findings revealed colitis in 7 (33%), adenoma in 4 (19%), hyperplastic polyps in 4 (19%), adenocarcinoma in 2 (9%), lymphoid aggregates in 2 (9%), melanosis coli in 1 (5%), and normal in 1 (5%) in the abnormal examination group. Abnormal endoscopic examination was found in 65.6% of patients. The prevalence of colitis, adenomas, and malignancy was high, therefore abnormal CRWT warrants further endoscopic evaluation.
Abstract: We have shown previously that 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3 (Gemini) compounds, which have two side chains attached to carbon-20, had increased anti-tumor activities against breast, prostate and leukemia cell lines in comparison to 1,25(OH)2 vitamin D3. This prompted us to synthesize additional Gemini compounds with further modifications and evaluate their anticancer effects. Most effective in this series was 1,25-dihydroxy-20S-21(3-hydroxy-3-methyl-butyl)-23-yne-26,27-hexafluoro-vitamin D3 [Gemini-23-yne-26,27-hexafluoro-D3]. This analog was approximately 10-fold more potent than previously characterized Gemini compounds in inhibiting the clonal growth of HL-60, MCF-7 and LNCaP cell lines. Also in MCF-7 cells, Gemini-23-yne-26,27-hexafluoro-D3 caused dephosphorylation of the oncogenic kinase, Akt, resulting in dephosphorylation of the Akt target proteins, Forkhead transcription factor and mammalian target of rapamycin (mTOR). Downstream effectors of mTOR were also inhibited by the analog as demonstrated by decreased phosphorylation of both S6 kinase, and the translation inhibitor, 4E-BP1. The mTOR pathway regulates mRNA translation; exposure of MCF-7 cells to Gemini-23-yne-26,27-hexafluoro-D3 decreased their rate of protein synthesis and increased the association of 4EBP-1 with the translation initiation factor, eIF4E. Inhibition of the Akt-mTOR pathway represents a novel mechanism by which vitamin D3 analogs may modulate the expression and activity of proteins involved in cancer cell proliferation.
Abstract: Differential display PCR analysis (DD-PCR) was used to identify novel genes that respond to IGF-I treatment in human MCF-7 breast cancer cells. Fifty-three cDNAs showed alterations in their mRNA levels in IGF-I treated cells. One of these genes showed a significant increase in the mRNA level in IGF-I treated cells in comparison to non-treated cells. We named this gene HIRF1 (human IGF-I regulated factor 1). Nucleotide blast analysis revealed that this gene has a 100% sequence identity with the sequence for BTG1 (B-cell translocation gene) binding factor 1 (human CCR4-associated factor 1 gene, hCAF1). By alignment of cloned HIRF1 cDNA and genomic DNA 8p21.3-p22 sequence, we were able to determine the exon-intron structure of the cloned HIRF1 gene on chromosome 8. Northern blot and real-time PCR analysis showed that BTG1 and c-fos reached their maximal expression fairly early within 10 min to 1 h, and decreased to basal levels after 3 h of IGF-I treatment. HIRF1/hCAF1 expression reached maximal stimulation after 3 h of IGF-I treatment and then gradually decreased to basal level. HIRF1 and BTG1 mRNA was inhibited by inhibitors of the cell signaling pathways, PI3/Akt kinase and MAPK kinases (ERK1/2 and p38). In summary, cloned HIRF1/hCAF1 is coregulated with BTG1 in response to IGF-I. The regulation of these genes as early response genes may have an important role in differentiation, growth and proliferation of breast cancer cells.
Abstract: PURPOSE: CCAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that regulate proliferation and differentiation in a variety of tissues. The purpose of this study was to explore the possibility that C/EBPalpha is involved in breast cancer. EXPERIMENTAL DESIGN: We quantified C/EBPalpha mRNA expression levels in 24 primary breast tumors, 16 normal breast samples, and 8 breast cancer cell lines using quantitative real-time reverse transcription-PCR assay. C/EBPalpha protein levels were further determined by immunohistochemical analysis. To examine the consequence of C/EPBalpha expression in breast cancer, we stably transfected an inducible C/EPBalpha expression vector into three breast cancer cell lines. RESULTS: Low expression of C/EBPalpha mRNA was found in 83% of primary breast cancer samples. Immunohistochemical study further showed either a markedly reduced or undetectable expression of C/EBPalpha protein in 30% of breast cancer specimens. The other 70% of breast cancers had C/EBPalpha expression in both the cytoplasm and nucleus; in control, C/EBPalpha was localized to the nucleus in the normal ductal cells. C/EBPalpha expression was associated with estrogen- and progesterone receptor-negative status. Induction of C/EBPalpha expression in these cell lines resulted in growth inhibition accompanied by G0-G1 cell cycle arrest and reduced anchorage-independent cell growth. C/EBPalpha expression was associated with down-regulation of c-myc and up-regulation of p21, PPARgamma, and the breast epithelial differentiation marker, maspin. CONCLUSIONS: These results suggest that reduced expression of C/EBPalpha may play a role in the development and/or progression of breast cancer.
Abstract: Mechanisms of estrogen-induced tumorigenesis in the target organ are not well understood. It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogens plays a critical role in estrogen-induced carcinogenesis. We tested this hypothesis by using an estrogen-induced hamster renal tumor model, a well established animal model of hormonal carcinogenesis. Hamsters were implanted with 17beta-estradiol (betaE2), 17alpha-estradiol (alphaE2), 17alpha-ethinylestradiol (alphaEE), menadione, a combination of alphaE2 and alphaEE, or a combination of alphaEE and menadione for 7 months. The group treated with betaE2 developed target organ specific kidney tumors. The kidneys of hamsters treated with alphaE2, alphaEE, or menadione alone did not show any gross evidence of tumor. Kidneys of hamsters treated with a combination of alphaE2 and alphaEE showed early signs of proliferation in the interstitial cells. Kidneys of hamsters treated with a combination of menadione and alphaEE showed foci of tumor with congested tubules and atrophic glomeruli. betaE2-treated tumor-bearing kidneys showed >2-fold increase in 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) levels compared with untreated controls. Kidneys of hamsters treated with a combination of menadione and alphaEE showed increased 8-iso-PGF(2alpha) levels compared with untreated controls, whereas no increase in 8-iso-PGF(2alpha) was detected in kidneys of alphaEE-treated group. A chemical known to produce oxidative stress or a potent estrogen with poor ability to produce oxidative stress, were nontumorigenic in hamsters, when given as single agents, but induced renal tumors, when given together. Thus, these data provide evidence that oxidant stress plays a crucial role in estrogen-induced carcinogenesis.
Abstract: Vascular endothelial growth factor (VEGF) has been shown to induce angiogenesis in vivo and in vitro. However, the association of plasma VEGF with tumor histopathology in high risk groups such as African American and non-white Hispanic women with breast cancer is not well understood. There is limited information on the prognostic relevance of plasma VEGF in patients who have had surgery and adjuvant treatment for breast cancer. In this study, we measured plasma VEGF from 125 minority women with primary breast tumor removal and were completing adjuvant treatment. The control group consisted of 20 subjects without cancer. We examined the association between plasma VEGF and other tumor characteristics such as steroid hormone receptors, tumor size, regional nodes, stage, recurrence, and overall survival. Our results confirmed that plasma VEGF levels were significantly higher in breast cancer patients than normal subjects. Plasma VEGF level increased in patients with increase in tumor size, and at late stage III/IV disease. Univariate analysis showed plasma VEGF to be a significant predictor of overall survival (RR=2.5, p=0.02). Multivariate analysis showed plasma VEGF not only to be an independent predictor of overall survival (RR=4.6, p=0.02) but also of local recurrence (RR=6.0, p=0.04). Tamoxifen in combination with CMF or CAF can reduce plasma VEGF level in patients with estrogen receptor positive tumor but not in estrogen receptor negative tumor. Our findings suggest that plasma VEGF should be considered as a tumor marker for breast cancer progression, and inhibitors of angiogenesis should be factored into the treatment protocol for patients who demonstrate increase in plasma VEGF levels at any stage of the disease.
Abstract: The transport of amino acids across the plasma membrane is a process of fundamental physiological importance. If this process is modified by estrogens and if estrogen receptors play a role in this regulation, then the alteration of metabolic events will be of significant importance in cancer cells which have high estrogen receptor content because estrogens modify cellular physiology through transactivational effects of their cognate receptors. In the present study, we investigated the role of 17 beta-estradiol on the regulation of different amino acid transport systems, in particular, Systems A, ASC and y+ in estrogen receptor positive (MCF-7, T47D, H-301) and estrogen receptor negative (MCF-10, SKBR-3, MDA-MB-231) cell lines. The cells were treated with 17 beta-estradiol (10 nM), ICI 182780 (1 microM), or 17 beta-estradiol plus ICI 182780. We discovered that 17 beta-estradiol specifically stimulates System A activity by 2- to 4-fold in estrogen receptor positive cell lines with a maximal stimulation 48 h after estrogen-treatment while no stimulation was observed in estrogen receptor negative cell lines. Estrogen-dependent activation of System A was inhibited by co-treatment with the antiestrogen ICI 182780. Northern blot analysis showed that System A mRNA levels are also increased following estrogen treatment, and this induction of mRNA transcript levels by estrogen can be inhibited by co-treatment with antiestrogen ICI 182780. The increase in System A transport activity following estrogen treatment was abbrogated when estrogen receptor positive cells were stably transfected with human antisense ER alpha cDNA. Kinetic analysis demonstrated that estrogen-induced stimulation results in a doubling of Vmax with no changes in Km. However, 17 beta-estradiol did not stimulate the activation of transport systems responsible for the transport of arginine (y+) or threonine (ASC). In summary, we have provided evidence that estrogen receptors play a role in the activation of System A by estrogen. This adaptation may have important physiological and nutritional significance on estrogen dependent growth of breast tumors.
Abstract: Amino acid starvation is a pathophysiological condition that results in protein deprivation due to cancer cachexia. Using the method of differential display of reverse transcription PCR (DDRT-PCR), we isolated a cDNA fragment in MCF-7 human breast cancer cells in response to amino acid starvation, which was identical with human CD24 gene. Northern blot results showed that CD24 mRNA in MCF-7 cells was constitutively expressed and significantly upregulated upon amino acid starvation. This stimulation was time-dependent and the maximal response was at 24 h. The expression of the amino acid starvation-induced CD24 mRNA decreased when starved cells were returned to a medium supplemented with amino acids. This repressive response was also time-dependent. Amino acid starvation-induced CD24 mRNA expression in MCF-7 cells was completely blocked by actinomycin D, which suggested that the regulation of CD24 mRNA by amino acid availability occurred at transcriptional level. When amino acid-starved cells were refed with amino acids for 8 h, the expression of CD24 mRNA declined to the basal levels confirming that CD24 mRNA expression could be stimulated by amino acid starvation. Interestingly, CD24 mRNA was poorly detected in MCF-10 cells, a benign human breast epithelial cell line. In conclusion, CD24 mRNA expression in MCF-7 cells was upregulated upon amino acid starvation. This amino acid starvation-induced upregulation of CD24 mRNA occurred at transcriptional level. The regulation of CD24 mRNA in MCF-7 cells by amino acid availability may play an important role in the progression and metastasis of human breast cancer.
Abstract: An estimated 7% of all breast cancers and 10% of all ovarian cancers are associated with inherited mutations in BRCA1 and BRCA2 genes. The mutations of a breast cancer-susceptible gene, BRCA1, confers increased risk of breast cancer in young women. Numerous studies have reported specific mutations in the BRCA1 and BRCA2 genes in the white population. However, there are very few studies on African-American and other ethnic minority groups. The goal of this study is to identify whether African-American patients with breast cancer carry some common mutations reported in other ethnic groups and whether they carry some novel mutations. We screened hot-region mutations on exons 2, 5, 11, 16, and 20 of BRCA1 gene in 54 African-American patients with breast cancer by NIRCA and SSCP methods. Our data revealed one novel frameshift mutation (3331 insG) and three missense sequence variants (A3537G, A3667G, and C4009T) on exon 11. Each sequence change was confirmed by automatic DNA sequencing. One rare sequence variant, A3537G, has been revealed in high frequency (3/54). Our data suggested that African-American patients with breast cancer carry some unique BRCA1 gene mutations.
Abstract: Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).
Abstract: The mutations in the breast cancer susceptible gene BRCA1 are responsible for about 50% of inherited breast cancers and confer increased risk of breast and ovarian cancer to its carriers. BRCA1 gene mutations may also be related with other types of cancers such as prostate cancer and colorectal cancer. The goal of this study was to investigate if BRCA1 mutation could be detected in diverse types of cancers. We used PCR-NIRCA and PCR-SSCP methods for screening the BRCA1 mutation hot regions, exons 2, 5, 11, 16 and 20. The positive samples were sequenced to confirm the nature of the mutations. We have identified a rare sequence variant, A3537G (Ser 1140Gly) in a B cell lymphoma patient and two polymorphisms, A1186G (Gln356Arg) in a brain cancer patient and A3667G (Lys1183Arg) in a germline tumor patient. In conclusion, 3 missense alterations of BRCA1 gene have been identified in cancers other than breast cancer.
Abstract: The anti-neoplastic properties of an Selenium compound were studied in vitro on several tumor cell lines: Breast (MCF-7, MCF-10, SKBR-3, BCAP37), Lung (RH2), Prostate (LNCap and PC-3), Colon (T84, Caco-2), Small Intestine (HCF8), and Liver (HepG2). We also examined additive or synergistic effect of Selenium in combination with standard anti-cancer drugs, Adriamycin (Doxorubicin) and Taxol. The effect of Selenium was assessed by apoptosis; DNA synthesis; growth rate by MTT assay; uptake of amino acid MeAIB by System A; and morphological changes. Our results demonstrate that MCF-7 and SKBR-3 showed increase in apoptosis as measured by DNA fragmentation and increase in "rounded" cells and membrane "blebbing", decrease in MeAIB uptake, and decrease in DNA synthesis. These changes were Selenium dose dependent with optimal inhibition at Selenium concentration between 4 and 40 ng/ml after 72 hrs of treatment. Similar observations were made with RH2, HCF8, Caco-2, and HepG2 cells. In contrast, LNCap, PC-3, and T-84 were not significantly affected by Selenium. However, addition of Adriamycin or Taxol in combination with Selenium caused small but significant inhibition of prostate cancer cells LNCap and PC-3. Addition of chemotherapeutic agents either Taxol or Doxorubicin with Selenium caused further inhibition of MCF-7, SKBR-3, RH2, HCF8, and HepG2 cells. In conclusion, Selenium has a significant anti-neoplastic effect on breast, lung, liver, and small intestinal tumor cells. Supplementation of Selenium enhanced chemotherapeutic effect of Taxol and Doxorubicin in these cells beyond that seen with the chemotherapeutic drugs used alone. These in vitro studies on several cancer cell lines suggest a potential benefit of Selenium-enhancement of anticancer effects other systems, and therefore offer further relevance to clinical trials efforts.
Abstract: We examined the significance of plasma HER-2/neu as a clinical or biological marker for assessing the progression of breast cancer in African American and Hispanic women with similar socioeconomic status, similar health insurance, and similar access to health care delivery. Base line studies show the following: average age of our breast cancer patients was 48 for Hispanic and 53 for African American women. Most of our patients presented invasive ductal carcinoma, and there was no ethnic difference. A larger number of Hispanic women had stage III/IV disease at the time of diagnosis. There was no significant difference in the number of African American or Hispanic patients with ER positive or negative receptors. However, a larger number of Hispanic women had PR positive tumors, and a larger number of African American women had PR negative tumors. In general, there was no difference in the levels of HER-2/neu between the two ethnic groups. Patients with tumors >5 cm had elevated plasma HER-2/neu. However, there was no ethnic difference between tumor size and HER-2/neu levels. In addition, regional node status had no impact on plasma HER-2/neu. Patients with stage III/IV tumors had elevated plasma HER-2/neu. No ethnic difference was observed at either stage I/II or III/IV. ER positive or negative status had no significant impact on plasma HER-2/neu in either ethnic group. In contrast, PR positive patients showed elevated plasma HER-2/neu. Plasma HER-2/neu (>60 U/ml) was the strongest predictor of overall survival, visceral site metastasis, and local recurrence.
Abstract: Rats and humans with chronic renal failure (CRF) are reported to have resistance to recombinant human insulin-like growth factor-1 (rhIGF-1). Because basal cytosolic calcium ([Ca2+]i), a second messenger, may be increased in CRF, this study was conducted to examine whether elevated basal [Ca2+]i may cause resistance to IGF-1. Cardiomyocytes from four groups of rats were studied: untreated CRF, CRF with parathyroidectomy (PTX), CRF with the calcium channel blocker felodipine (F), and sham operation of the kidney (SO). CRF was created by ligation of two-thirds of the left renal artery and contralateral nephrectomy. Rats from each group were pair-fed the same diet for 20 to 22 d. Basal [Ca2+]i in cardiomyocytes (nM) in the CRF rats (102.0 +/- 2.8; SEM), was significantly higher than in each of the CRF-PTX, CRF-F, and SO groups (65.2 +/- 1.9, 63.8 +/- 2.6, and 63.5 +/- 2.0, respectively; P < 0.01). rhIGF-1 increased cardiomyocyte [Ca2+]i in all four groups of rats. The rise in [Ca2+]i was significantly diminished in the CRF rats (P < 0.05) and did not differ among the CRF-PTX, CRF-F, and SO rats. Protein synthesis after incubation with 0, 50, 100, 200, or 400 ng/ml rhIGF-1 was lower in cardiomyocytes from CRF rats than in each of the other three groups (P < 0.05) and was significantly less in the CRF-F rats compared with SO animals. IGF-1 receptor mRNA and IGF-1 receptor number and affinity were not different among the four groups. These findings suggest that cardiomyocytes from CRF rats display elevated basal [Ca2+]i and attenuated rhIGF-1-induced increase in [Ca2+]i; basal protein synthesis is decreased, and IGF-1-stimulated protein synthesis is impaired; elevated basal [Ca2+]i seems to contribute to this diminished response to rhIGF-1.
Abstract: Breast cancer is a leading cancer in American women. About 7% of breast cancer is due to inheritance of mutated genes BRCA1 and BRCA2. Numerous investigations have revealed a number of mutations in BRCA1 and BRCA2 genes. The inheritance of the mutated BRCA1 or BRCA2 genes accounts for 45% and 35%, respectively, of hereditary breast cancers. A central database named Breast Cancer Information Core (BIC) has been established in the National Human Genome Research Institute (NHGRI) to coordinate the information related with BRCA1 and BRCA2 research. Nearly half of the mutations (49%) in the BRCA1 gene are frameshift mutations and the cancer-causing mutations account for 66% of all entries. However, for the BRCA2 gene frameshift mutations and cancer-causing mutations account for only 35% and 43%, respectively, of all entries. The significance of a large portion of missense sequence variants (24% of BRCA1 mutations and 47% of BRCA2 mutations) needs further evaluation. The incidence of 185delAG and 5382insC in BRCA1 gene and 6174delT in BRCA2 gene is predominantly high and the founder effect of these mutations is discussed.
Abstract: In vitro studies have shown that insulin-like growth factor (IGF) is a mitogen for breast cancer cells. However, the associations of plasma IGF-I with tumor histopathology in high-risk groups need further investigation. We hypothesize that plasma IGF-I and serum IGFBP3 concentrations in breast cancer patients may provide useful information on the progression of their disease, and determine the probability of recurrence and survival. We have carried out a retrospective study on 130 minority breast cancer patients. Plasma IGF-I and serum IGFBP3 were correlated with tumor histopathology, menopausal status, treatment modality, recurrence rates, and probability of survival. Plasma IGF-I and serum IGFBP3 were measured by radioimmunoassay. Our studies show that breast cancer patients have elevated plasma IGF-I and serum IGFBP3 levels. In addition we observed the following: IGF-I did not correlate with age and nodal stage. IGF-I and IGFBP3 increased with tumor size (T4). IGF-I did not correlate with estrogen receptor status, but did increase in progesterone-receptor-positive patients. IGF-I levels were higher in premenopausal patients and in women with cancer recurrence. Tamoxifen reduced IGF-I levels significantly and reduced the risk of recurrence. The survival probability was greater in patients with plasma IGF-I levels <120 ng/ml. In conclusion, lowering of plasma IGF-I may offer the following benefits: (a) reduce the risk of developing breast cancer in high-risk groups; (b) slow the progression of breast cancer in patients at early stages of cancer; (c) lower the risk of recurrence, and (d) increase the probability of survival. Copyright Copyright 1999 S. Karger AG, Basel
Abstract: Hyperlipidemia is an established risk factor for progressive renal damage and proteinuria. Platelets and vascular smooth muscle cells (VSMC) are known to possess low density lipoprotein (LDL) cholesterol receptors. We used platelet LDL receptors to investigate the hypothesis that elevated lipids could activate intracellular calcium [Ca2+]i signals, leading to altered vascular tone and permeability. We divided essential hypertensives into microalbuminuric positive (MA+) and negative (MA-) groups and measured baseline and LDL stimulated levels of [Ca2+]i. The MA+ group demonstrated a significantly higher mean baseline [Ca2+]i level (119.0 +/-24.5 v 86.2 +/- 25.4 micromol/mL, P = .001). The MA+ group also displayed greater elevations in [Ca2+]i levels after stimulation with LDL in concentrations of 10 and 25 microg/mL (100.9 +/- 54.8 v 40.9 +/- 20.2, P = .04 and 111.6 +/- 51.0 v 52.9 +/- 39.9 micromol/mL, P = .03, respectively). Our data indicate that hypertensive patients with early glomerular capillary injury display exaggerated influx of [Ca2+]i after LDL receptor stimulation. Heightened LDL receptor sensitivity may facilitate LDL mediated [Ca2+]i signals, leading to increased VSMC tone and proliferation and progressive renal disease.
Abstract: The intracellular accumulation of albumin has been observed in cytosols of benign and malignant human breast tumors and in mammary tumors of rodents induced by carcinogens. Additionally, cellular uptake of albumin has been detected in MCF-7 human breast cancer cells in culture. The clinical relevance of the albumin accumulation in human and rodent mammary tumors is not clear. In this study, we investigated the accumulation of albumin in an estrogen-induced and -dependent hamster kidney tumor model to understand the mechanisms and the role of hormones in this process. Protein accumulation patterns were examined by Western blot analyses in kidney homogenates of hamsters treated with 17beta-estradiol for various lengths of time and in kidney tumors which are induced with 100% incidence by this treatment for at least six months. Such analyses were also carried out in tissues of hamsters treated with the weakly carcinogenic estrogen 17alpha-ethinylestradiol (10% tumor incidence after nine months of treatment). Our data demonstrate the accumulation of albumin in kidney of hamsters treated with 17beta-estradiol but not with 17alpha-ethinylestradiol. Albumin accumulates specifically in the target organ of carcinogenesis, the kidney, however, with no increase in the serum concentrations or in the liver. Tumors do not develop in the livers of hamsters under these conditions of 17beta-estradiol treatment. This accumulation of albumin in hamster kidney may be the result of damage to the glomerulum which may be compromised by estradiol-induced toxicity and therefore unable to filter out excess albumin.
Abstract: The actions of insulin-like growth factor 1 (IGF-1) on protein turnover and of the IGF-1 receptor (IGF-1R) were examined in skeletal muscle of rats with chronic renal failure (CRF) and sham operated (SO), pair-fed controls. Acidemia was prevented in CRF rats with NaHCO3. Serum IGF-1 and skeletal muscle IGF-1 and IGF-1 mRNA were reduced in CRF rats. Dose-response studies revealed impaired stimulation of protein synthesis and suppressed inhibition of protein degradation by IGF-1 in epitrochlearis muscle of CRF rats. Neither IGF-1 analogues with low affinity to IGF binding proteins nor proteinase inhibitors obliterated the IGF-1 resistance. In CRF rats, skeletal muscle IGF-1R mRNA was increased; displacement ligand binding studies and affinity labeling of the IGF-1R alpha subunit indicated increased total skeletal muscle IGF-1R number with normal affinity. However, both autophosphorylation of the IGF-1R beta subunit (i.e., IGF-1R tyrosine kinase) and the IGF-1R tyrosine kinase activity towards exogenous insulin receptor substrate-1, a natural substrate for IGF-1R tyrosine kinase, were reduced in CRF fats. These data indicate that in skeletal muscle of CRF rats there is resistance to the IGF-1 effects on protein synthesis and degradation and decreased IGF-1 and IGF-1 mRNA levels; IGF-1R mRNA and number are increased; but activity of IGF-1R tyrosine kinase is impaired. This postreceptor defect may be a cause of the skeletal muscle resistance to IGF-1 in CRF.
Abstract: BACKGROUND: A rat model of acute renal failure (ARF) with sepsis (ARF+S) was developed to simulate the clinical syndrome of hypercatabolic illness in patients with ARF. METHODS: Sepsis was created by ligation and needle puncture of the cecum; ARF was created by left renal artery clamping and contralateral nephrectomy. RESULTS: Two studies were performed. In study 1, rats with sham surgery, sepsis, ARF, and ARF+S were examined for 48 hours. During the first 24 hours after surgery, ARF and ARF+S rats displayed increased urea and ammonia nitrogen appearances and abnormal plasma amino acid levels. These abnormalities were exaggerated in ARF+S rats. In study 2, sham, ARF, and ARF+S rats were injected with sodium bicarbonate or normal saline. During the first 24 hours after surgery, the ARF and ARF+S rats showed an increase in urea nitrogen appearance to 210% and 293%, respectively, of sham values, which was greater than the levels that have been previously reported. Sodium bicarbonate treatment did not influence nitrogen output. CONCLUSIONS: Rats with ARF+S may be a useful model for studying catabolic patients with ARF. The lack of effect of sodium bicarbonate on nitrogen balance merits additional study.
Abstract: The amino acid L-citrulline is an important intermediate of urea cycle and a key precursor for arginine biosynthesis. We have examined the characteristics of citrulline transport across the everted sacs of the rat small intestine. Our studies suggest that the optimal site of citrulline absorption is middle to lower ileum. It shows active transport, and this transport is predominantly Na+ dependent. Its uptake is significantly inhibited by ouabain, dinitrophenol, sodium azide, and sodium cyanide. Kinetic estimation reveal an apparent substrate concentration at 1/2 maximum velocity of 4.10 +/- 0.86 mM and a Vmax of 18.7 +/- 1.66 mumol/g wet weight tissue/30 min. Analog inhibition studies suggest that citrulline may share the neutral brush border system described for the mucosal brush border membranes of the rabbit jejunum or a system analogous to system ASC described for nonepithelial cells and for basolateral membranes of certain epithelia. In conclusion, the rat small intestine has developed a specific carrier-mediated, Na(+)-dependent pathway for citrulline absorption.
Abstract: The human erythroleukemic cell K-562 serves as an in vitro model to study changes in cell surface antigens and mechanisms regulating globin gene expression associated with in vivo erythropoiesis. In this report we have examined the regulation of amino acid transport systems, in particular, systems A and ASC, during differentiation of erythroleukemic cells. For additional comparison we examined the uptake of leucine, 3-aminoendobicyclo-(3,2,1)-octane-3-carboxylic acid (BCO), arginine, and glutamate. Hexamethylene-bis-acetamide (HMBA), dimethyl sulfoxide, and butyrate induce cell differentiation with a block in G1-G0 phase of the cell cycle. These agents caused a significant downregulation of 2-(methylamino)isobutyric acid uptake by system A. In contrast, the Na(+)-dependent threonine uptake by system ASC remained unaltered. The uptake of leucine, BCO, arginine, and glutamate by as yet unidentified systems was, however, stimulated after HMBA treatment. Hemin, a potent inducer of hemoglobin synthesis in K-562 cells, does not block cell cycle events and, interestingly, had no significant effect on both systems A and ASC. These differences in inducer actions suggest that system A activity may be related to specific stages of cell differentiation and perhaps to other cellular signals.
Abstract: Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na(+)-independent, with an overshoot between 5-10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1 = 31 +/- 1.82 and Km2 greater than 198 +/- 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37 degrees C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane beta-alanine carrier system, with inhibition by beta-alanine, hypotaurine, and taurine. alpha-Alanine, 2-methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-taurine [N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+ gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.
Abstract: The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.
Abstract: We have studied the changes in amino acid transport in fetal erythroid cells isolated from rat fetal liver at different gestation days. Our results show that System A transport as measured by the Na+-dependent uptake of 2-(methylamino)isobutyric acid (MeAIB) was conspicuous at day 13 but virtually disappeared between days 16 and 18. In contrast, the activity of System ASC measured by the Na+-dependent uptake of MeAIB-insensitive threonine uptake increased after day 14 and was optimal between days 16 and 18. This transport system regressed in activity with further maturation, but remained conspicuously saturable in the matured red blood cell. Interestingly, the newly discovered Na+-independent System asc (Vadgama, J. V., and Christensen, H.N. (1985) J. Biol. Chem. 260, 2912-2921), selective for the uptake of test substrates threonine, serine, and alanine, was present in these erythroid cells. Its activity increased during gestation days 16-18. System L transport was present simultaneously with the Na+-independent System asc. As we had previously demonstrated for the pigeon red blood cell, these two transport systems are kinetically independent as confirmed with inhibition studies and the special selectivity of System L to trans stimulation. Tryptophan uptake could be attributed predominantly to System L, as also observed for the nucleated pigeon red blood cells and certain other cells. Arginine showed its familiar Na+-independent mode of uptake as a cation throughout the interval of study. An exceptional Na+-dependent component of arginine uptake emerged after day 14, peaked at day 18, and then disappeared on further maturation of the erythroid cell.
Abstract: On the basis of inhibition analysis two bicyclic amino acid analogs appear to enter human red blood cells by much the same Na+-independent mediation, whereas striking differences are apparent in the routes for tryptophan and leucine, confirming a role for System T, but also suggesting the participation of a third system of low affinity somewhat selective for weakly basic amino acids. System T of the human cell is specifically inhibited by 4-azidophenylalanine, and is highly sensitive, relative to System L, to N-ethylmaleimide inhibition. Uptake by System T approaches its steady state much more slowly than does System L, and its participation in trans-stimulation is questionable, whereas that of System L is as usual strong. A different added transport system became apparent in the slow approach of the Na+-independent mediation of uptake of 3- and 4-carbon dipolar amino acids by the nucleated pigeon red cell to its steady state. In that cell System T makes at most a minor contribution. The patterns of trans-stimulation of fluxes among selected pairs of amino acids in the pigeon cell correspond to a usual participation in transmembrane exchange by System L, and also by the new transport system. An important but not the sole source of the heterogeneity in the pigeon cell is the participation of the system conspicuously involved in the transport of alanine, serine, and threonine, among other amino acids. This route of transport of these amino acids is made conspicuous by their small transport by other Na+-independent agencies, notably System L. Reactivity with this system is enhanced by a side change hydroxyl or sulfhydryl group. Uptake by this route as tested by threonine showed little inhibition by cysteinesulfinate under conditions inhibitory to System asc; also a sensitivity to lowering of pH unlike that seen with System asc. The new Na+-dependent transport system appears to be a species variant of quite similar Na+-independent systems discovered by Young et al. (Young, J. D., Ellory, J. C., and Tucker, E. M. (1975) Nature (Lond.) 254, 156-157; Fincham, D. A., Mason, D. K., and Young, J. D. (1982) Biochem. Soc. Trans. 11, 776-777) in sheep and horse erythrocytes on the basis of their absence in phenotypes. These authors have emphasized several similarities in these two cases to Na+-dependent System asc, and they propose that Na+ dependence has specifically been lost on maturation of the red cells without major changes in amino acid selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)
Abstract: Criteria have been set up for recognizing the trait of interconvertibility of transport System ASC, whereby it operates in a deprotonated form to mediate the transport of zwitterionic amino acids and in a protonated form to transport anionic amino acids. This trait has been detected by each criterion applied, in all the tested occurrences of System ASC, as follows: the Ehrlich ascites tumor cell in suspension; a cultured variant thereof in monolayer; the Chinese hamster ovary cell CHO-K1 in monolayer; the pigeon red blood cell in suspension; and the human red blood cell in suspension. We conclude that this trait is a general feature of System ASC which may we may use provisionally in defining the system.
Abstract: 1. Transport of L-homocitrulline, an amino acid which occurs in milk products, was studied with rat small intestine in vitro and from the human mouth in vivo. Absorption was partially dependent, in both systems, on the presence of sodium ions. 2. Metabolic inhibitors decreased L-homocitrulline uptake across the small intestine. Transport across the intestine did not occur against the concentration gradient but did show saturation kinetics. 3. The barbiturate, amytal, did not inhibit buccal absorption. Saturation kinetics were demonstrated. 4. Experiments were conducted with L-citrulline, or other amino acids, as possible inhibitors of L-homocitrulline transport. Results were compatible with Na+-dependent carrier-mediated uptake across the buccal mucosa. Active transport could be involved with the small intestine assuming that L-homocitrulline has a low affinity for the carrier system.
Abstract: In contrast to the changes seen in membrane transport systems for other neutral, anionic, and cationic amino acids, System N for glutamine, histidine, and asparagine in the rat hepatocytes shows nearly constant properties at the fetal, differentiated, and cultured hepatoma stages. These properties were tested by measuring the Na+-dependent transport of glutamine. This approximate constancy applies not only to the transport selectivity of the system among neutral amino acids, but also to its tolerance of Li+ as a substitute for Na+, its characteristic sensitivity to pH lowering, its relative sensitivity to N-ethylmaleimide, its stimulation by amino acid deprivation, and its failure to respond to insulin or glucagon. The properties of histidine as a substrate for System N were also examined. Inhibition studies with different cell types suggest that the Na+-dependent glutamine and histidine uptake is more restricted to System N in the hepatoma line H35 (H4-11-EC,3) and in the fetal hepatocyte than in hepatoma line HTC and the Ehrlich cells. The Na+-independent component of glutamine and histidine uptake was greater in the hepatoma cells in continuous culture than in fetal and adult hepatocytes in primary culture. Trans-stimulation of glutamine and histidine influx into H35 cells occurs predominantly by the Na+-independent route.
Abstract: The isomeric 3-aminobicyclo[3.2.1]octane-3-carboxylic acids were synthesized and compared with the widely used (1R,2S,4S)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid as to specificity to the Na+-independent membrane transport system L of the Ehrlich ascites tumor cell and of the rat hepatoma cell line HTC. The presence of an additional methylene group in the ring system leads to an optically symmetrical amino acid, with the advantages that the product is devoid of isomeric contamination. Hence, optical resolution is not necessary to secure a homogeneous test substrate for discrimination of amino acid transport systems. Through its inhibitory action on the cellular uptake of known system-specific amino acids, the bicyclo[3.2.1]octane amino acid proved more reactive than the bicycloheptane analogue with the Na+-independent amino acid transport system of the test cells and not perceptibly reactive with the accompanying Na+-dependent systems. Recent evidence of the presence of a second component of Na+-independent amino acid transport, beyond system L, increases the importance of securing a variety of possibly discriminatory model substrates.
