(68)Ga-AMBA and (18)F-FDG for preclinical PET imaging of breast cancer: effect of tamoxifen treatment on tracer uptake by tumor.

INTRODUCTION: AMBA is a bombesin analogue that binds to GRPr. In a mouse model of estrogen-dependent human breast cancer, we tested whether (68)Ga-AMBA can be used for PET detection of GRPr-expressing tumors and could be more accurate than (18)F-FDG to monitor tumor response to hormone therapy. METHODS: The radiolabeling of (68)Ga-AMBA was automated using a R&D Synchrom module. ZR75-1, a breast cancer cell line, was xenografted in nude mice. (68)Ga-AMBA tumor uptake was compared with that of (18)F-FDG before and after treatment with tamoxifen. RESULTS: AMBA was (68)Ga-radiolabelled in 30min with 95.3% yield and purity≥98%. Prior to treatment, (68)Ga-AMBA was highly concentrated into tumors (tumor to non-tumor ratio=2.4 vs. 1.3 with (18)F-FDG). With tamoxifen treatment (n=6) (68)Ga-AMBA uptake plateaued after 1week and decreased after 2weeks, with a significant reduction compared to controls (n=4). In contrast the effect of tamoxifen treatment could not be appreciated using (18)F-FDG. CONCLUSIONS: (68)Ga-AMBA appeared better than (18)F-FDG to visualize and monitor the response to hormone treatment in this breast cancer model.

A germ-line p53 mutation accelerates pulmonary tumorigenesis: p53-independent efficacy of chemopreventive agents green tea or dexamethasone/myo-inositol and chemotherapeutic agents taxol or adriamycin.

Recent evidence indicates that individuals with a p53 germ-line mutation (Li-Fraumeni syndrome) have a 50% risk of developing lung cancer by age 60. In this study, p53 heterozygous knockout mice and p53 transgenic mice carrying a dominant negative mutant were crossed with the A/J mouse, which is highly susceptible to lung tumor induction, to investigate whether a p53 germ-line mutation is a predisposing gene for carcinogen-induced pulmonary adenomas in mice. The number of lung tumors was not significantly increased in (TSG-p53 x A/J)F1 p53 heterozygous knockout mice as compared with that in (TSG-p53 x A/J)F1 wt mice 16 weeks after exposure to N-nitrosomethylurea (MNU). In contrast, an average of 22 lung tumors were observed in (UL53-3 x A/J)F1 mice carrying a mutant p53 transgene (135Valp53) compared with an average of 7 lung tumors seen in (UL53-3 x A/J)F1 wt mice after treatment with N-nitrosomethylurea. Similar enhancement of lung tumor multiplicity (approximately 3-fold) was seen when mutant versus wt mice were treated with the tobacco-related carcinogens benzo[a]pyrene or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. These results suggest that the mutant p53 transgene may have a dominant negative effect on the wt p53. The potential usefulness of this new mouse model in lung cancer chemoprevention and chemotherapy was examined. The chemopreventive efficacy of the green tea or a combination of dietary dexamethasone and myoinositol and the chemotherapeutic efficacy of Taxol or Adriamycin was examined in wt mice or mice with a mutation in the p53 gene. Mice treated with dexamethasone/myo-inositol and green tea displayed an average of 70 and 50% inhibition of lung tumors, respectively, regardless of p53 status. Similarly, when mice bearing established lung adenomas were treated with Taxol or Adriamycin, a decrease in tumor volume of approximately 70% was observed independent of p53 mutation status. Thus, the (UL53-3 x A/J)F1 p53 transgenic mouse seems to be an excellent model for human carriers of p53 germ-line mutations (Li-Fraumeni syndrome). Furthermore, the lung adenomas generated in this model possess mutations in both the K-ras proto-oncogene and the p53 tumor suppressor gene. This model should prove directly useful for chemoprevention and chemotherapy studies.

Chemopreventive efficacy of promising farnesyltransferase inhibitors.

The studies presented were designed to test the efficacy of farnesyltransferase inhibitors (FTIs) as potential chemopreventive compounds in the mouse lung tumor model, and in tumor cell lines. The compounds included manumycin, gliotoxin, dihydroepiandrosterone (DHEA), perillyl alcohol (POH), and FTI-276. Each of these compounds had the potential, based on in vitro and limited in vivo evidence, to inhibit mouse lung tumorigenesis. In vitro studies were conducted with both K-ras-transformed NIH-3T3 cells and mouse lung tumor epithelial cell lines. We utilized 2 primary mouse lung tumor models that reliably produce lung tumors with an oncogenic K-ras mutation when induded by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Manumycin, gliotoxin, DHEA, and POH were administered 3 times per week peritoneally (i.p.), starting 1 week prior to carcinogen treatment, and throughout the test period (4.5 months). FTI-276 was delivered daily for 4 months by a time-release pellet method. Both the manumycin and gliotoxin treatment groups demonstrated 100% incidence and an increase in tumor multiplicity over control, of 66% and 58% increase respectively (P < .05). Although DHEA showed no significant chemopreventive effect, POH treatment demonstrated a 22% reduction in tumor incidence (P < .05) and a 58% reduction in tumor multiplicity (P < .05). Finally, FTI-276 reduced both the tumor multiplicity by 41.7% (P < .005), and the total tumor volume/burden per mouse by 79.4% (P < .0001). The apoptotic index in FTI-276-treated tumors showed an increase of 77% over control tumors (P < .05). In vitro, all compounds demonstrated growth inhibition at a dose-response manner; however, manumycin, gliotoxin, and DHEA demonstrated an initial increase in growth rate at lower doses. In summary, we have shown that POH and FTI-276 are chemopreventive in a primary mouse lung tumor model. In contrast, DHEA was not significantly chemopreventive at the dosage utilized, and treatment of an immunocompetent host with manumycin or gliotoxin demonstrated a significant increase in tumorigenicity over carcinogen control.

Effect of farnesyltransferase inhibitor FTI-276 on established lung adenomas from A/J mice induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The Ras protein undergoes a series of post-translational modifications at the C-terminal CAAX motif, which culminates with the anchoring of p21 Ras to the plasma membrane where it relays growth regulatory signals from receptor tyrosine kinases to various pathways of cell signal transduction. FTI-276 is a CAAX peptidomimetic of the carboxyl terminal of Ras proteins. Pharmacokinetic analysis of FTI-276 in A/J mice with a time-release pellet system showed a dose of 50 mg/kg body wt achieved an average serum level of 1.68 microg/ml for up to 30 days following implantation. In the present study, 4 week old A/J mice were initiated with a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (100 mg/kg), and monitored for 18 weeks. Mice were grouped for daily delivery (time-release pellet) of 50 mg/kg of FTI-276 for 30 days (n = 12) and the control group (n = 12). Analysis of tumors from time-release pellet treated animals showed a 60% reduction in tumor multiplicity and a 42% reduction in tumor incidence. Moreover, FTI-276 treatment resulted in a significant reduction in tumor volume (approximately 58%). Mutation analysis of the lung tumors from both treatment groups revealed that most of the tumors harbored mutations in the codon 12 of K-ras and there is no significant difference in the incidence and types of mutations between tumors from the treated and control animals. This is the first demonstration of chemotherapeutic efficacy of a synthetic CAAX peptidomimetic farnesyltransferase inhibitor in a primary lung tumor model.

Cdkn2a encodes functional variation of p16INK4a but not p19ARF, which confers selection in mouse lung tumorigenesis.

The cyclin-dependent kinase inhibitor 2a (Cdkn2a) locus encodes two distinct tumor suppressors, p16INK4a and p19ARF, whose functions interrelate in the regulation of cell proliferation as key components of the retinoblastoma and p53 pathways, respectively. In many types of cancer, alterations of Cdkn2a abrogate the functions of both suppressors, implying that both are integral to the genesis of certain cancer types. While this has been observed in mouse lung adenocarcinogenesis, recent observations also suggested that naturally occurring variation at the Cdkn2a locus is probably operative in the development of these tumors. Firstly, two common haplotypes of mouse Cdkn2a have been identified, each of which encodes cosegregating variants of p16INK4a and p19ARF. The p16INK4a variants differ at amino acids 18 (histidine or proline) and 51 (valine or isoleucine), whereas the p19ARF variants differ only at amino acid 72 (histidine or arginine). Secondly, genetic resistance to lung tumor formation appears to segregate with one particular haplotype, which also is deleted preferentially in lung adenocarcinomas of Cdkn2a heterozygous mice. Here we attempt to explain these observations and to characterize further the roles of p16INK4 and p19ARF in mouse lung tumorigenesis by examining the function and expression of each of the variants of Cdkn2a. Functional analysis showed that the proline 18/isoleucine 51 p16INK4a variant was diminished in cdk6 binding, cdk6 inhibition and NIH/3T3 fibroblast growth suppression compared with the histidine 18/valine 51 variant, whereas both of the p19ARF variants suppressed growth with similar potencies. Also, the different alleles for p16INK4a and p19ARF were transcribed equally in the normal lungs of Cdkn2a heterozygotes, as determined by comparative reverse transcription-polymerase chain reaction-single-stranded conformation polymorphism analysis. These results indicate that strain-specific variation in p16INK4a function is exploited in mouse lung tumorigenesis and strongly implicate a role for p16INK4a in lung cancer predisposition and development.

5-Aza-2'-deoxycytidine is chemopreventive in a 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone-induced primary mouse lung tumor model.

Carcinogenesis is a multistep process in which many alterations in both genetic and epigenetic controls lead to a growth advantage for neoplastic cells. Hypermethylation has been established as the basis of genomic imprinting, but recent studies have also shown that alterations in genomic methylation patterns may contribute to tumorigenesis. The chemical 5-aza-2'-deoxycytidine (5-aza-dC) has been used both in vitro and in vivo to inhibit DNA methylation. In this study, we investigated the chemopreventive efficacy of 5-aza-dC in a well-established primary mouse lung tumor model. Five-week-old male (C3H/HeJ x A/J) F1 hybrid mice were treated for 24 consecutive weeks with 5-aza-dC, three times per week i.p. Lung tumors were induced with two consecutive weekly doses of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone starting 1 week after initial treatment with 5-aza-dC. We demonstrated that 5-aza-dC exhibits a chemopreventive effect in this primary mouse lung tumor model which, like human lung adenocarcinomas, harbors an activating K-ras mutation. Treatment with 5-aza-dC resulted in a 23% reduction in tumor incidence, as well as a 42% reduction in tumor multiplicity. This work supports further investigation of methylation inhibitors likes 5-aza-dC for early intervention, prevention and treatment of lung cancer.

Cytotoxicity of tocopherols and their quinones in drug-sensitive and multidrug-resistant leukemia cells.

Cytotoxicities of tocopherols (alpha-T, gamma-T, delta-t), their para (alpha-TQ, gamma-TQ, delta-TQ)- and ortho (Tocored)-quinone oxidation products, the synthetic quinone analog of gamma-TQ containing a methyl group substituted for the phytyl side-chain (TMCQ) and the synthetic quinone analog of Tocored containing a methyl group substituted for the phytyl side-chain (PR) were measured in acute lymphoblastic leukemia cell lines that are drug-sensitive (CEM) and multidrug-resistant (CEM/VLB100). Among tocopherols, only delta-T exhibited cytotoxicity. Among para quinones, alpha-TQ showed no cytotoxicity, while gamma-TQ and delta-TQ were highly cytotoxic in both CEM and CEM/VLB100 cell lines (LD50 < 10 muM). delta-TQ and gamma-TQ were more cytotoxic than the widely studied chemotherapeutic agent doxorubicin, which also showed selective cytotoxicity to CEM cells. The orthoquinone Tocored was less cytotoxic than doxorubicin in drug-sensitive cells but more cytotoxic than doxorubicin in multidrug-resistant cells. Cytotoxicity was not a function of the phytyl side-chain since both TMCQ and PR were cytotoxic in leukemia cells. Cytotoxic para and ortho quinones were electrophiles that formed adducts with nucleophilic thiol groups in glutathione and 2-mercaptoethanol. Cytotoxicity was enhanced when the glutathione pool was depleted by preincubation with buthionine-[S,R]-sulfoximine, but cytotoxicity was diminished by the addition of N-acetylcysteine to cultures. alpha-T also diminished the cytotoxicity of para- and orthoquinones. Buthionine-[S,R]-sulfoximine did not block the inhibitory effect of either N-acetylcysteine or alpha-T, showing that these agents did not act solely by maintaining the glutathione pool as an essential antioxidant system. In conclusion, tocopherylquinones represent a new class of alkylating electrophilic quinones that function as highly cytotoxic agents and escape multidrug resistance in acute lymphoblastic leukemia cell lines.

Chemopreventive effect of perillyl alcohol on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced tumorigenesis in (C3H/HeJ X A/J)F1 mouse lung.

This study was designed to test the chemopreventive potential of perillyl alcohol, an inhibitor of farnesyltransferase, in a mouse lung tumor bioassay. Perillyl alcohol is a naturally occurring monoterpene found in lavender, cherries, and mint. We have shown previously that the majority of lung tumors in this bioassay have an activating mutation in the K-ras gene, which occurs early in the development of mouse lung carcinogenesis. The Ras protein undergoes a series of post-translational modifications, the first of which is farnesylation at the cysteine of the C-terminal CAAX motif. These modifications lead to the anchoring of Ras p21 to the plasma membrane in its biologically active state. Activated Ras p21 couples growth regulatory signals from receptor tyrosine kinases to cytoplasmic second messengers. In a preliminary study, we determined the maximum tolerated dose of perillyl alcohol to be 75 mg/kg body weight. For the bioassay, 5-week-old male (C3H/HeJ X A/J) F1 hybrid mice were randomized into trial groups, and treated with perillyl alcohol three times per week i.p., starting 1 week prior to initiation with the carcinogen NNK, and continuing for 22 weeks after initiation. Our results show a 22% reduction in tumor incidence, and a 58% reduction in tumor multiplicity. Our study demonstrates that perillyl alcohol is an effective chemopreventive compound in the mouse lung tumor bioassay.

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts.

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11: EC 3.4.24.11) and aminopeptidase N (APN: EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV: EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC50 = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC50 = 8 microM), and APN by amastatin (IC50 = 50 nM) and bestatin (IC50 = 100 microM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.

Thyronines and probucol inhibition of human capillary endothelial cell-induced low density lipoprotein oxidation.

Oxidized lipoproteins have been implicated as important factors in the pathogenicity of atherosclerosis. Thus, antioxidants play a significant role in inhibiting a critical step in atheroma progression. Previously, we demonstrated that thyronine analogs inhibit Cu(2+)-induced low density lipoprotein (LDL) oxidation. In the present study, we examined the effect of thyronine analogs on endothelial cell (EC)-induced LDL oxidation. LDL was incubated with or without EC in the presence or absence of various concentrations of thyronine, vitamin C, or probucol at 37 degrees in a humidified atmosphere (95% air, 5% CO2). Thyronine analogs, probucol, and vitamin C inhibited EC-induced LDL oxidation in a concentration-dependent manner. The concentration of each agent (microM) producing 50% inhibition (IC50) of EC-induced LDL oxidation for thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility, respectively, was as follows: 0.294 and 0.417 for levothyroxine (L-T4); 0.200 and 0.299 for L-triiodothyronine (L-T3); 0.125 and 0.264 for dextro-thyroxine (D-T4); 0.203 and 0.304 for reversed triiodothyronine (rT3); 1.02 and 1.44 for probucol; and 13.6 and 14.9 for vitamin C. Thyroid binding globulin (TBG) inhibited EC-induced LDL oxidation; further, thyronines bound to TBG exhibited more antioxidant activity than unbound thyronines. Pretreatment of EC with any of the thyronines decreased the ability of EC to oxidize LDL. Also, our results showed that a synergistic interaction exists between vitamin C and T4 in the inhibition of EC-induced LDL oxidation. The T4 and TBG concentrations that inhibited LDL oxidation were in the physiological range. We conclude that T4, like the pharmacological agent probucol, reduces oxidative modification of LDL and thus may act as a natural inhibitor of atherogenesis.

Correlation between AIDS-related Kaposi sarcoma histological grade and in vitro behavior: reduced exogenous growth factor requirements for isolates from high grade lesions.

Kaposi sarcoma, the most common AIDS-associated malignancy, affects 10-30% of all AIDS patients. To date, research into the biological characteristics of AIDS-related Kaposi sarcoma (AIDS-KS) derived cell lines has been based on cultures established from skin explants or pleural effusions/peritoneal fluids. We have established several AIDS-KS lines from biopsy confirmed oral mucosal and epidermal AIDS-KS lesions and have found a correlation between AIDS-KS lesional grade and in vitro cellular growth characteristics. In comparison to epidermal AIDS-KS lesions, mucosal AIDS-KS lesions frequently possessed both a more advanced histologic grade and demonstrated a greater capacity to proliferate in minimal medium. We report the ability of AIDS-KS isolates from high grade lesions to sustain proliferation (greater than 60 population doubling levels) in medium not supplemented with endothelial cell growth supplement and/or cytokine rich conditioned medium. These findings indicate that AIDS-KS cells isolated from high grade lesions have reduced requirements for exogenously provided growth supplements, and suggest that increased autologous cytokine production accompanies AIDS-KS lesional progression.

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts.

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III. and bradykinin (BK) and converted ANG I to the active metabolite ANG(1-7). ANG III was converted to the novel ANG IV metabolite [des-Arg1]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.

Human microvascular endothelial cell-extracellular matrix interaction in cellular growth state determination.

We introduce two methods, both of which are based on cellular-extracellular matrix interaction, which will facilitate the study of human microvascular endothelial cells. One method describes the means to obtain a G1 population baseline in human microvascular endothelial cells. Because of the contribution of the extracellular matrix in endothelial cell growth, synchronization in G1 was possible only after the incorporation of angiostatic levels of heparin and hydrocortisone into the extracellular matrix. In the second method, we demonstrate that selective perturbation of human microvascular endothelial cell-extracellular matrix interactions results in the induction of a transitional growth state, between proliferative and differentiated growth states, in human microvascular endothelial cells. In the functional, microtubule formation assays, transitional growth state endothelial cells display rates that are indermediate between those obtained from differentiated and proliferative endothelial cells. Our results demonstrate the importance of the human microvascular endothelial cell-extracellular matrix interaction in the determination of cellular growth state. Our findings also imply that responsiveness of microvascular endothelial cells to their cellular-extracellular matrix environs is highest during the differentiated growth state.

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities.

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained inflammatory states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levels of virus-induced cytokines and, because of a sparing effect on their phagocytic cells, retention of the phagocytic inflammatory response. A component of phagocytic activation is the initiation of the oxidative burst, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess decreased cytoprotective capabilities. Relative to either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplemented with conditioned medium from MOT, an HTLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed an inherent biochemical responsiveness, by increasing catalase activity following exposure to exogenous H2O2. In contrast, the catalase activity of AIDS-KS cells decreased following H2O2 challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression of AIDS-KS.

Environmental induction of tumor phenotype in a putative Kaposi sarcoma progenitor cell.

Many features of AIDS-related Kaposi sarcoma (AIDS-KS), e.g., multifocal lesional presentation at sites perfused by the microvasculature, suggest that AIDS-KS is initially a hyperplasia that subsequently progresses to a neoplasia. We propose that the unique AIDS environment, which contains high levels of circulating factors such as viral cytokines, is key in initiating the KS lesion. Further, we maintain that due to their physiological function, human microvascular endothelial cells (HMECs) are both likely target cells for the AIDS-related cytokines, and are putative AIDS-KS progenitor cells. Previously, we have shown that as a component of HMEC transition between proliferative and differentiated growth, HMECs modulate their nucleotide and glutathione levels. After attaining contact inhibition, HMECs enter a state of differentiation, which is characterized by cellular entrance into a G0, quiescent growth state, a decrease in cellular bioenergetic profiles, and spontaneous formation of microtubules. In contrast, when cultured in a "KS milieu", HMECs fail to differentiate. Instead, the "KS milieu" cultured cells assume a "growth relaxed" phenotype and demonstrate a lack of contact inhibition, loss of anchorage dependence, and retention of a "proliferative" bioenergetic profile despite culture confluence. Our results imply both that HMECs are responsive to AIDS-related cytokines, and that the local environment is key to instigating a relaxation of cellular growth controls.

Modulation of human microvascular endothelial cell bioenergetic status and glutathione levels during proliferative and differentiated growth.

During angiogenesis, formerly differentiated human microvascular endothelial cells (HMECs) return to a proliferative growth state. Many fundamental questions regarding HMEC function, such as how HMECs adapt to changes in bioenergetic requirements upon return to proliferative growth, remained unanswered. In this study, we evaluated whether modifications in HMEC bioenergetic profiles and glutathione (GSH) levels accompanied the cellular transition between differentiated and proliferative growth. To provide insight into the continuum of cellular adaptations that occur during this transition, we used a method recently developed in our laboratory that induces a state of morphological and functional predifferentiation in HMECs. Cellular morphology, in conjunction with flow cytometric DNA analyses and HMEC functional assays (the directed migration and intercellular association involved in microtubule formation) were employed to validate the HMEC culture state of growth. Analysis of the HPLC nucleotide profiles disclosed several findings common to all culture growth states. These uniform findings, e.g., cellular energy charges > 0.90, and highly reduced redox states, revealed that cultured HMECs maintain high rates of oxidative metabolism. However, there were also significant, culture growth state related differences in the nucleotide profiles. Proliferative HMECs were shown to possess significantly higher (relative to both large vessel endothelial cells, and differentiated HMECs) levels of GSH and specific nucleotides which were related with a return to the active cell cycle-ATP, GTP, UTP, and CTP, and NADPH. Further, the nucleotide profiles and GSH levels of the predifferentiated HMECs were determined to be intermediate between levels obtained for the proliferative and differentiated HMECs. The results of this study demonstrate that the capacity to modulate their cellular bioenergetic status during growth state transitions is one of the adaptations that enable HMECs to retain a growth state reciprocity. In addition, our findings also show that HMECs, especially during the proliferative growth state, are biochemically distinct from endothelial cells harvested from large vessels, and therefore suggest that HMECs are the cells of choice to employ when studying diseases that affect the human microvasculature.