Evaluation of an affordable HIV-1 virological failure assay and antiretroviral drug resistance genotyping protocol.

HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDR(ultralight)). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS EasyQ v1.2, Roche) with an R(2) of 0.99. Accuracy studies illustrated standard deviations of <1 log RNA copies/ml for plasma and DBS ARTA-VFA results compared to the reference method. The ARTA-VFA's intended use was to deliver qualitative results either < or >5000 RNA copies/ml. No significant differences in the proportion of results < or > either the 5000 RNA copies/ml or 1000 RNA copies/ml cut-off were noted for plasma indicating either cut-off to be useful. Significant differences were noted in these proportions when DBS were used (P=0.0002), where a 5000 RNA copies/ml cut-off was deemed more appropriate. The sensitivity and specificity of the ARTA-VFA with plasma were 95% and 93% and 91% and 95% for DBS using a 5000 RNA copies/ml cut-off. The ARTA HIVDR(ultralight) assay was reliable for plasma and DBS samples with a viral load >5000 RNA copies/ml, with amplification and sequencing success rates of 91% and 92% respectively for plasma, and 95% and 80% respectively for DBS. HIVDR profiles for plasma and DBS were 100% concordant with the reference assay. This study evaluated a previously described combination of two assays potentially useful in assessing HIV-1 virological failure and resistance, showing good concordance with reference assays. These assays are simple to perform and are affordable, viable options to detect virological failures in certain resource limited settings. The assays' compatibility with DBS sampling extends the access of HIV-1 virological monitoring to more remote settings.

Differential induction of hepatic estrogen receptor and vitellogenin gene transcription in Xenopus laevis.

Low levels of estrogen receptor (200-500 per cell) are present in the liver of hormonally naive male Xenopus. However, administration of estradiol results in a rapid 2- to 5-fold increase in cellular estrogen receptor content concurrent with the de novo transcriptional activation of the genes for the yolk protein precursor vitellogenin. Studies on Xenopus embryogenesis suggest that estrogen receptor induction is required for the activation of vitellogenin transcription. The purpose of the present study was to examine the mechanism of estrogen receptor induction in male Xenopus liver. The experimental protocol used 4-hydroxytamoxifen, an antiestrogen with a high affinity for the estrogen receptor, to inhibit the effects of estradiol. Changes in estrogen receptor content were then determined through the use of an exchange assay. 4-Hydroxytamoxifen alone suppressed the level of estrogen receptor from 800 sites per cell in hormonally naive animals to 250 sites per cell. Administration of estradiol 24 h after the antiestrogen resulted in the induction of estrogen receptor to a level equivalent to that found in control animals (800 sites per cell). However, under the same conditions, estradiol was unable to overcome the antiestrogen inhibition of vitellogenin gene transcription. Although 4-hydroxytamoxifen displayed a high affinity for the hepatic estrogen receptor, it did not inhibit the binding of estradiol to a middle affinity cytoplasmic estrogen-binding protein. These results suggest that different mechanisms are involved in the induction of estrogen receptor and vitellogenin gene transcription.

Posttranscriptional regulation of albumin gene expression in Xenopus liver: evidence for an estrogen receptor-dependent mechanism.

We have previously shown that estrogen administration to male Xenopus laevis results in the posttranscriptional suppression of serum albumin mRNA concurrent with the transcriptional activation of the genes for the yolk protein precursor vitellogenin. To determine whether the posttranscriptional regulation of albumin gene expression is mediated through a mechanism involving the high affinity estrogen receptor protein or through a receptor-independent mechanism involving a middle affinity cytoplasmic estrogen-binding protein we examined the effects of the competitive estrogen receptor antagonist 4-hydroxytamoxifen. Administration of 4-hydroxytamoxifen 24 h before estradiol completely blocked both the suppression of albumin mRNA and the transcriptional activation of the vitellogenin genes. Albumin gene transcription remained constitutive under all treatment regimens. Competitive binding experiments demonstrated that 4-hydroxytamoxifen has an affinity for the estrogen receptor similar to that of estradiol. However, 4-hydroxytamoxifen displays little or no interaction with the middle affinity cytoplasmic estrogen-binding protein. These data indicate that the estrogen receptor occupies a key role in the posttranscriptional regulation of albumin mRNA.

Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells.

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.

Effect of estrogens and antiestrogens on growth-regulatory enzymes in human breast cancer cells in tissue culture.

The effect of estrogens and antiestrogens is examined on three enzymes the activities of which are known to correlate with cell growth. Estrogen treatment increases thymidylate synthetase binding sites up to 4-fold over controls. The extent of induction is dependent on incubation time and the basal rate of cell growth in untreated cells. Amount of active enzyme generally shows a positive correlation with rates of DNA synthesis and cell growth. Thymidine kinase activity and the number of dihydrofolate reductase binding sites are similarly induced by estrogen treatment. Conversely, the effect of antiestrogens on MCF-7 cells is exceedingly complex in that responses in enzyme activities and several generally accepted indices of cell growth (cell number, protein content, rate of DNA synthesis) are dissimilar. Dose response, magnitude of response, and direction of response (increase or decrease) are distinct for each enzyme and for each measure of cell growth with each antiestrogen tested. These results suggest that specific cellular activities are modulated independently by estrogens and antiestrogens and that changes in ligand-receptor complex cannot be the sole explanation for the specificity of estrogen and antiestrogen action. Some degree of specificity and heterogeneity may reside at the level of receptor interaction with the various genes subject to estrogenic modulation.

Hormonal regulation of de novo pyrimidine synthesis and utilization in human breast cancer cells in tissue culture.

The hormonal regulation of pyrimidine synthesis and utilization is examined in the estrogen-responsive MCF-7 human breast cancer cell line. [14C]Acetate is used as a novel marker in assaying the incorporation of pyrimidine nucleotides derived through de novo synthesis into DNA. Technical verification of the validity of this tracer in DNA synthesis is presented. The experimental conditions permitting extrapolation from radioactivity incorporated into DNA to pmol of DNA synthesized/unit of time are discussed. We observe that estrogen administration increases the incorporation of [14C]acetate into DNA, and this effect is most evident after 36 hr of exposure to hormone. Treatment with the antiestrogen tamoxifen produces substantial reductions in the rate of incorporation of [14C]acetate into DNA. Incorporation correlates well with net rates of DNA synthesis, as measured by 32Pi incorporation into DNA. Concomitant administration of thymidine in concentrations in excess of 10(-7)m partially relieves tamoxifen inhibition of net DNA synthesis and increases the apparent stimulation of net DNA synthesis following 18 to 32 hr of estrogen treatment. Activity of de novo enzymes such as carbamylphosphate synthetase, aspartate transcarbamylase, orotidine pyrophosphorylase, and orotidine decarboxylase also varies with hormone treatment. All characteristically increase following estrogen treatment and decrease after tamoxifen administration. These results suggest that changes in de novo pyrimidine synthesis after hormone treatment are directly linked to hormone-induced alterations in the activity of specific-enzymes in the de novo pathway.

Dihydrofolate reductase gene amplification and possible rearrangement in estrogen-responsive methotrexate-resistant human breast cancer cells.

Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5' flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.

Hormonal regulation of net DNA synthesis in MCF-7 human breast cancer cells in tissue culture.

Estrogens and anti-estrogens have striking effects on growth of target tissues in vivo. In order to examine these effects in vitro, experimental methods for the rigorous assessment of rates of net DNA synthesis in tissue culture systems are required. The quantitation of DNA synthesis with inorganic [32P]orthophosphate is described. The limitations of more conventional [3H]thymidine labeling are illustrated in this hormonally responsive system. Estrogen administration increases the specific activity of acid-soluble phosphate and increases net DNA synthesis relative to controls in MCF-7 human breast cancer cells. Stimulation is most evident after 36 hr of hormone treatment. Conversely, tamoxifen produces substantial inhibition of net DNA synthesis after 36 hr of hormone treatment. Thymidine availability modulates the effect of estrogens and anti-estrogens on DNA synthesis and constitutes an independent experimental variable.

The functional properties and innervation density of type II mechanoreceptor units of the sural nerve of the rabbit.

The functional properties of Type II mechanoreceptor units of the sural nerve of the rabbit have been examined, using controlled mechanical stimuli applied to the skin. The response of Type II units exhibited 5 phases: (1) a resting spontaneous phase (2) an open phase, (3) a transient adaptive phase, (4) a regular static phase, and (5) an off phase. The impulse frequency/indentation depth relationship for the static phase was best fitted by a linear function with a slope of 0.015 and an intercept of 2.47. An almost as good fit could be obtained with a power function (exponent of 0.57 and a multiplying constant of 17.2). The impulse frequency/velocity characteristics was best fitted by a power function; it became horizontal at an average value of 5 micrometer/s. The mean threshold if Type II units was 17.2 micrometer; there was no change in response frequency once the displacement reached 1340 micrometer. There are approximately 206 Type II units innervating a skin area of 59.1 cm2, giving an innervation density of 3.42. The significance of the similarities and differences between Type II units in the rabbit compared with those in the cat are discussed.

The spatial distribution and functional properties of type I mechanoreceptor units of the sural nerve of the rabbit.

The functional characteristics and spatial arrangement of Type I mechanosensitive units found in the sural nerve of the anaesthetized rabbit have been investigated. There are approximately 108 Type I units innervating a skin area of about 59.1 cm2 supplied by the sural nerve giving an innervation density of 1.83. This innervation density of 1.83 does not vary over the skin territory innervated by the sural. The dome/fibre ratio was 2.7. The domes were found to be distributed in a dispersed but fairly regular pattern. The average nearest neighbour distance was 5.3 mm. A stimulus of 5.5 threshold units showed the existence of a small receptive field of radius 2 mm surrounding each dome; at a 3 mm radius even the largest 2 mm indentation failed to evoke a response. The response of the Type I units exhibited 4 distinct phases: (1) a dynamic phase; (2) a transient adaptive phase; (3) a highly irregular static phase; (4) an off phase. The impulse frequency/static displacement relationship was best fitted by a power function. The impulse frequency-velocity characteristic became horizontal at an average value of 15 micrometers/ms. A power function provided the best fit for this relationship. The possible physiological significance of the differences between properties of Type I receptor populations in the rabbit and cat are discussed.

The responses of dorsal horn neurones to controlled mechanical stimulation of the hindlimb skin in the rabbit.

Seven types of neurones responding to mechanical stimulation of the hindlimb skin were identified in the dorsal horn of the spinal cord of the rabbit. Of the 218 units examined, 33% were activated by movement of hairs--H cells; 25% were excited by stimulation of Type I domes--on/off cells; 15.6% were spontaneously active and were excited by vertical displacement of the skin and lateral skin stretch--SPE (spontaneous excited) cells; 4.4% were spontaneously active and were inhibited by mechanical stimulation of the skin--SPI (spontaneous inhibited) cells; 5.2% were spontaneously active and responded to application of pressure--SPEP (spontaneous excited pressure) cells; 3% were spontaneously active and were excited by movement of joints--SPEJ (spontaneous excited joint) and 13.8% of neurones responded to hair movement and also to some other forms of mechanical stimulus--CM (combined mechanosensitive) cells. Of the 218 units examined, 133 were located in lamina IV and 85 in lamina V. A quantitative analysis of some of the characteristics of the on/off, SPE and SPI cells is carried out and a model of the dorsal horn system activated by Type I and Type II receptor units is proposed.

Association between steroid hormone receptor status and disease-free interval in breast cancer.

The possibility of an association between steroid hormone receptor status and disease-free interval was examined in 292 patients with breast cancer. Estrogen receptor positivity was associated with a prolonged disease-free interval. This association was independent of age, menopausal status, tumor size, or nodal status. There was no association between the presence or absence of progesterone, androgen, or glucocorticoid receptor and disease-free interval.

Relationship between the progesterone, androgen, and glucocorticoid receptor and response rate to endocrine therapy in metastatic breast cancer.

PIP: 85 patients with metastatic or surgically unrecsectable primary breast cancer who had had 1 or more steroid hormone receptor assays performed immediately before the institution of endocrine therapy were studied retrospectively to determine any influence of steroid hormone receptors on response rate to endocrine therapy. Included in addition to effects of estrogen receptor (ER) status are the effects of androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) on therapy perfornamce. Of 18 patients whose tumor contained PR, 11 responded to endocrine therapy, compared with 8 of 26 patients with low or absent PR. PR increased the predictive index of the ER in an group of patients who had received no prior therapy, but it did not help in patients who had received prior endocrine therapy. 0 of 4 patients whose tumors were ER negative but PR positive responded to endocrine therapy. Present trends suggest a possible association between AR and GR and response to endocrine therapy. A cut-off value of 10 fmol/mg of cytoplasmic protein was needed to make these trends apparent. The distributions of AR and GR values for responders and nonresponders were not significantly different. Knowledge of AR status does not increase the protective index in ER-positive or ER-negative tumors. GR positivity may increase the predictive index in ER-positive tumors, but not in ER-negative ones.^ieng

Distribution, frequency, and quantitative analysis of estrogen, progesterone, androgen, and glucocorticoid receptors in human breast cancer.

The distribution and frequency of steroid hormone receptors are described 329 patients with breast cancer. The distribution of each of the steroid hormone receptors is unimodal with a progressive increase in the proportion of patients positive at lower receptor values. Receptor values expressed as fmol/mg cytoplasmic protein are well correlated with values expressed as fmol/mg breast tumor. Estrogen receptor was positive in 53% of the patients; progesterone receptor was positive in 38% of the patients; glucocorticoid receptor was positive in 52% of the patients; and androgen receptor was positive in 31% of the patients. The type of tissue assayed did not affect steroid hormone receptor positivity. For primary tumors, there was no correlation between steroid hormone receptor positivity and location of the tumor in the breast, size of the tumor, or extent of the disease. Each of the steroid hormone receptors was positively associated with each of the other steroid hormone receptors. Estrogen receptor was correlated with menopausal status and axillary nodal status, but these correlations did not exist for the other steroid hormone receptors. Estrogen receptor was not correlated with age after adjustment for menopausal status. The other steroid hormone receptors were not correlated with age.

PIP: This study describes the distribution and frequency of estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), and glucocorticoid receptor (GR) in a large series of patients with primary metastatic breast cancer. 329 patients were in this series. All 4 steroid hormone receptors were present in the population: ER was positive in 53%, PR was positive in 38%, AR was positive for 31%, and GR was positive in 52%. Next, the distribution of ERs as well as the distributions of PR, AR, and GR values seemed unimodal. There was a very high correlation between any steroid hormone receptor value expressed as either fmol/mg of cytoplasmic protein or fmol/mg of breast tumor. Of more importance was that alternate methods of data expression did not alter the classification of values as positive or negative. No correlation was found between any of the steroid hormone receptors and laterality of the breast tumor, location and size of the primary tumor, extent of disease, or type of tissue assayed. None of the steroid hormone receptors correlated with age. There was a strong correlation noted between ER values and menopausal status. Neither PR, AR, nor GR was significantly associated with menopausal status. ER status was correlated with axillary nodal status, with the ER positive group containing a high proportion of node-negative patients. Finally, quantitative analysis of steroid receptor hormone values demonstrated correlations among other receptors. Plotting values of any 1 receptor vs. any other receptor resulted in a positive Kendall rank test correlation which was highly significant.

Association between steroid hormone receptors and response rate to cytotoxic chemotherapy in metastatic breast cancer.

The influence of steroid hormone receptors on response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer was determined in a retrospective study. We have previously reported that 34 of 45 patients with tumors containing low or absent estrogen-receptor values had objective responses to chemotherapy while three of 25 patients with positive estrogen-receptor tumors responded. In the present study, 22 of 34 patients with low or absent progesterone-receptor tumors had an objective response to cytotoxic chemotherapy, while none of eight patients with a positive progesterone-receptor tumor responded (P less than 0.05). Patients having tumors with a negative estrogen receptor and a negative progesterone receptor had a response rate of 88% (21 of 24 patients). There were three patients whose tumors were estrogen-receptor negative but progesterone-receptor positive; none had a response to chemotherapy. Chemotherapy response was not associated with the presence or absence of either androgen or glucocorticoid receptor. We conclude that progesterone-receptor values in addition to estrogen-receptor status may prove to be important correlates of response to cytotoxic chemotherapy in metastatic breast cancer. Androgen- and glucocorticoid-receptor analyses are not helpful in predicting response to chemotherapy.

The relation between estrogen receptors and response rate to cytotoxic chemotherapy in metastatic breast cancer.

In a retrospective study we determined the relation between estrogen receptors and the response rate to cytotoxic chemotherapy in 70 patients with metastatic breast cancer. Thirty-four of 45 patients with low or absent estrogen-receptor values (less than 10 fmol per milligram of cytoplasmic protein) had objective responses to chemotherapy, whereas only three of 25 patients with higher values (greater than 10 fmol per milligram of cytoplasmic protein) responded (P less than 0.0001). There were no statistically significant differences between the two groups in age, menopausal status, disease-free interval, Karnofsky index or prior therapy. Differences in sites of involvement or type of chemotherapy did not account for the increased response rate in receptor-negative patients. We conclude that estrogen-receptor values are an important predictor of response to cytotoxic chemotherapy in metastatic breast cancer.