Progesterone receptor isoform-dependent cross-talk between prolactin and fatty acid synthase in breast cancer.

Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes.

GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.

The many faces of prolactin in breast cancer.

Prolactin (PRL) is a neuroendocrine polypeptide hormone primarily produced by the lactotrophs in the anterior pituitary gland of all vertebrates. The physiological role of PRL in mammary glands is relatively certain while its role in breast tumor has been a topic of debate for over 20 years. In this review, the author attempts to briefly summarize the data coming from his laboratory in the past years, focusing on G129R, a PRL receptor (PRLR) antagonist developed by introducing a single amino acid substitution mutation into human PRL (hPRL) at position 129, and a variety of G129R derivatives. The author has proposed two novel ideas for potential use of PRL, not anti-PRL agents, as an adjuvant agent for breast cancer, making it a hormone of many faces.

Dynamic analysis of GH receptor conformational changes by split luciferase complementation.

The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharmacological manipulation, genetic alteration of JAK2 levels, and truncation of the GHR intracellular domain (ICD) tail suggested that GH acutely enhances proximity of the GHR homodimer partners independent of the presence of JAK2, phosphorylation of GHR-luciferase chimeras, or an intact ICD. However, subsequent reduction of complementation requires JAK2 kinase activity and the ICD tail. This conclusion is in contrast to existing models of the GHR activation process.

Antagonism of tumoral prolactin receptor promotes autophagy-related cell death.

Therapeutic upregulation of macroautophagy in cancer cells provides an alternative mechanism for cell death. Prolactin (PRL) and its receptor (PRLR) are considered attractive therapeutic targets because of their roles as growth factors in tumor growth and progression. We utilized G129R, an antagonist peptide of PRL, to block activity of the tumoral PRL/PRLR axis, which resulted in inhibition of tumor growth in orthotopic models of human ovarian cancer. Prolonged treatment with G129R induced the accumulation of redundant autolysosomes in 3D cancer spheroids, leading to a type II programmed cell death. This inducible autophagy was a noncanonical beclin-1-independent pathway and was sustained by an astrocytic phosphoprotein (PEA-15) and protein kinase C zeta interactome. Lower levels of tumoral PRL/PRLR in clinical samples were associated with longer patient survival. Our findings provide an understanding of the mechanisms of tumor growth inhibition through targeting PRL/PRLR and may have clinical implications.

The role of prolactin receptor in GH signaling in breast cancer cells.

GH and prolactin (PRL) are structurally related hormones that exert important effects in disparate target tissues. Their receptors (GHR and PRLR) reside in the cytokine receptor superfamily and share signaling pathways. In humans, GH binds both GHR and PRLR, whereas PRL binds only PRLR. Both hormones and their receptors may be relevant in certain human and rodent cancers, including breast cancer. GH and PRL promote signaling in human T47D breast cancer cells that express both GHR and PRLR. Furthermore, GHR and PRLR associate in a fashion augmented acutely by GH, even though GH primarily activates PRLR, rather than GHR, in these cells. To better understand PRLR's impact, we examined the effects of PRLR knockdown on GHR availability and GH sensitivity in T47D cells. T47D-ShPRLR cells, in which PRLR expression was reduced by stable short hairpin RNA (shRNA) expression, were compared with T47D-SCR control cells. PRLR knockdown decreased the rate of GHR proteolytic turnover, yielding GHR protein increase and ensuing sensitization of these cells to GHR signaling events including phosphorylation of GHR, Janus kinase 2, and signal transducer and activator of transcription 5 (STAT5). Unlike in T47D-SCR cells, acute GH signaling in T47D-ShPRLR cells was not blocked by the PRLR antagonist G129R but was inhibited by the GHR-specific antagonist, anti-GHR(ext-mAb). Thus, GH's use of GHR rather than PRLR was manifested when PRLR was reduced. In contrast to acute effects, GH incubation for 2 h or longer yielded diminished STAT5 phosphorylation in T47D-ShPRLR cells compared with T47D-SCR, a finding perhaps explained by markedly greater GH-induced GHR down-regulation in cells with diminished PRLR. However, when stimulated with repeated 1-h pulses of GH separated by 3-h washout periods to more faithfully mimic physiological GH pulsatility, T47D-ShPRLR cells exhibited greater transactivation of a STAT5-responsive luciferase reporter than did T47D-SCR cells. Our data suggest that PRLR's presence meaningfully affects GHR use in breast cancer cells.

Stromal-epithelial interactions modulate cross-talk between prolactin receptor and HER2/Neu in breast cancer.

Prolactin (PRL) promotes the proliferation and survival of breast cancer cells in part via the transactivation of human epidermal growth factor receptor 2 (HER2), also known as Neu in rodents. A PRL receptor (PRLR) antagonist, G129R, has been developed, which indirectly inhibits the tyrosine phosphorylation of HER2 (p-HER2) in human breast cancer cell lines. In this study, we investigate the effects of cancer-associated fibroblasts (CAFs) upon this molecular cross-talk using tumor cells and CAFs derived from spontaneous mammary tumors of female MMTV-neu transgenic mice. Tumors were resected and cultured as small tumor chunks (~3 mm3) or were cultured in monolayer. G129R reduced tyrosine phosphorylation of Neu (p-Neu) in a dose-dependent manner (IC50~10 µg/ml) in tumor chunks, but had no effect on primary tumor epithelial cells grown in monolayer. Direct co-culture of mouse or human tumor epithelial cell lines with CAFs restored the epithelial cells' response to G129R, similar to that observed in mouse tumor chunks. The addition of PRL, as expected, induced p-Neu in both the tumor chunk and co-culture models. The inhibitory effect of G129R was absent when CAFs were physically separated from mouse tumor epithelial cells using a transwell system, or when CAFs were replaced with normal fibroblasts in direct co-culture with human or mouse tumor epithelial cells. In vivo, G129R reduced p-Neu levels in primary mammary tumors of mice in a time- and dose-dependent manner. In conclusion, CAFs play a critical role in bridging the cross-talk between PRL and HER2/Neu in both mouse and human models of breast cancer. The inhibitory effects of G129R on p-Neu and on tumor growth are dependent upon interactions of tumor epithelial cells with CAFs.

Growth hormone signaling in human T47D breast cancer cells: potential role for a growth hormone receptor-prolactin receptor complex.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are structurally similar cytokine receptor superfamily members that are highly conserved among species. GH has growth-promoting and metabolic effects in various tissues in vertebrates, including humans. PRL is essential for regulation of lactation in mammals. Recent studies indicate that breast tissue bears GHR and PRLR and that both GH and PRL may impact development or behavior of breast cancer cells. An important facet of human GH (hGH) and human PRL (hPRL) biology is that although hPRL interacts only with hPRLR, hGH binds well to both hGHR and hPRLR. Presently, we investigated potential signaling effects of both hormones in the estrogen receptor- and progesterone receptor-positive human T47D breast cancer cell line. We found that this cell type expresses ample GHR and PRLR and responds well to both hGH and hPRL, as evidenced by activation of the Janus kinase 2/signal transducer and activator of transcription 5 pathway. Immunoprecipitation studies revealed specific GHR-PRLR association in these cells that was acutely enhanced by GH treatment. Although GH caused formation of disulfide-linked and chemically cross-linked GHR dimers in T47D cells, GH preferentially induced tyrosine phosphorylation of PRLR rather than GHR. Notably, both a GHR-specific ligand antagonist (B2036) and a GHR-specific antagonist monoclonal antibody (anti-GHR(ext-mAb)) failed to inhibit GH-induced signal transducer and activator of transcription 5 activation. In contrast, although the non-GHR-specific GH antagonist (G120R) and the PRL antagonist (G129R) individually only partially inhibited GH-induced activation, combined treatment with these two antagonists conferred greater inhibition than either alone. These data indicate that endogenous GHR and PRLR associate (possibly as a GHR-PRLR heterodimer) in human breast cancer cells and that GH signaling in these cells is largely mediated by the PRLR in the context of both PRLR-PRLR homodimers and GHR-PRLR heterodimers, broadening our understanding of how these related hormones and their related receptors may function in physiology and pathophysiology.

Improving the pharmacokinetics/pharmacodynamics of prolactin, GH, and their antagonists by fusion to a synthetic albumin-binding peptide.

To prolong the circulation half-life of human prolactin (hPRL), human GH (hGH), and their competitive antagonists, hPRL-G129R and hGH-G120R, we examined the effects of fusing a serum albumin-binding peptide (SA20) to their amino- or carboxyl-terminus. Fusion of the SA20 peptide to the amino-terminus of the ligands was less detrimental upon their ability to induce or inhibit signal transduction and cell proliferation in vitro than fusion to the carboxyl-terminus. Pharmacokinetic (PK) studies in mice revealed that the half-life of SA20-hPRL and SA20-hGH was prolonged and their clearance was reduced in comparison with hPRL and hGH. Pharmacodynamic (PD) studies in 8-week-old female mice revealed that lobuloalveolar development in mammary glands was greater in all three groups (daily, every 2 days, or every third day over a 12-day period) of mice treated with SA20-hPRL (4 mg/kg) compared with hPRL (3.59 mg/kg). Similarly, daily administration (i.p.) of SA20-hGH (8 mg/kg) or hGH (7.15 mg/kg) to 23-day-old female mice over a 40-day period revealed the superiority of SA20-hGH over hGH as measured by weight gain, body length, and lobuloalveolar development in the mammary glands. These findings indicate that SA20 modification of hPRL, hGH, and their respective antagonists improves their PK/PD properties.

Combination therapy using three novel prolactin receptor antagonist-based fusion proteins effectively inhibits tumor recurrence and metastasis in HER2/neu transgenic mice.

Previously, prolactin receptor antagonist (G129R)- based fusion proteins were developed including G129R fusions with an angiogenesis inhibitor (endostatin), an immune system modulator (interleukin 2), and a modified truncated cytotoxin (PE38KDEL). Each fusion protein was designed to target the PRLR-positive cells via the G129R moiety and at the same time attack a hallmark common to cancer cells via the second moiety. In this study, we tested the efficacy of the three fusion proteins as a combination therapy in an aggressive but clinically relevant mouse tumor model. To test the feasibility and to optimize a treatment regimen, allografts of a mammary carcinoma cell line (McNeuA) derived from an MMTV-neu transgenic mouse were first used. Growth of the allografts was significantly retarded by regimens which combined all three fusion proteins. In addition, a significant increase in cytotoxic CD8+ T cells was observed within the tumors of the combination treated groups. After establishing the dosing regimen, two doses of cocktail treatment (low and high doses administered twice weekly) along with individual component controls were administered to female MMTV-neu transgenic mice after surgical removal of a naturally occurring tumor. The average tumor recurrence time was significantly delayed in both low and high combination treatment groups in comparison to the no treatment control group (34, 50 and 18 days, respectively). The total number of lung metastases was also significantly decreased in both combination treatment groups. In conclusion, using G129R-based fusion proteins to target mammary carcinomas and to tackle multiple hallmarks of cancer at the same time was an effective strategy for treating HER2-postive mammary cancer in this mouse tumor model.

Rassf3 is responsible in part for resistance to mammary tumor development in neu transgenic mice.

MMTV/neu transgenic mouse line is a well-documented model for studying HER2/neu-related breast cancer. Approximately 80% of these mice develop mammary tumors by 11 months of age, whereas a small percentage appears to have naturally acquired resistance to HER2/neu tumorigenesis. To identify factors responsible for tumor resistance in these transgenic mice, comparative genetic profiling was used to screen alterations in gene expression in the mammary gland. A novel gene, the RAS association domain (RalGDS/AF-6) family 3 (Rassf3), which belongs to a family of RAS effectors and tumor suppressor genes, was identified. Data indicated 1) that Rassf3 is overexpressed in mammary gland of tumor-resistant MMTV/neu mice compared to tumor-susceptible MMTV/neu littermates or non-transgenic mice, and 2) Rassf3 is significantly up-regulated in neu-specific mouse mammary tumors compared to adjacent normal tissues. In vitro overexpression of RASSF3 inhibited cell proliferation in HER2/neu positive human and mouse breast cancer cell lines, possibly through induction of apoptosis. A novel MMTV/Rassf3-neu bi-transgenic mouse line, overexpressing Rassf3 and neu genes in mammary glands, was established. Mammary tumor incidence in bi-transgenic mice was delayed compared to their MMTV/neu+/- littermates. These data suggest that Rassf3 may influence mammary tumor incidence in MMTV/neu transgenic mice.

Activation of growth hormone receptors by growth hormone and growth hormone antagonist dimers: insights into receptor triggering.

GH binds dimerized GH receptors (GHRs) to form a trimolecular complex and induces downstream signaling events. The mechanism by which GH binding converts the inactive predimerized GHR to its active signaling conformation is uncertain. GH has no axis of symmetry. Its interaction with GHR is mediated by two asymmetric binding sites on GH, each with distinct affinity. Site 1 is of high affinity and is thought to mediate the first binding step. Mutation of binding site 2 (as in the human GH mutant, G120R) disrupts the second binding but leaves site 1 binding intact. G120R is a GH antagonist; it binds only one GHR and thus fails to signal, and it prevents productive GHR binding by normal GH. We previously demonstrated that prolactin receptor signaling was achieved by a dimeric version of a prolactin antagonist. We now employ assays of cellular signaling and receptor conformational changes to examine whether GH molecules harboring two site 1 regions can trigger GHR activation. We used recombinantly produced GH-GH and G120R-G120R dimers in which monomers in tandem are connected by a short linker peptide. Rabbit GHR-expressing human fibrosarcoma cells (C14) were treated with GH, G120R, GH-GH, or G120R-G120R. As expected, GH and GH-GH, but not G120R, induced GHR disulfide linkage, as assessed by anti-GHR blotting of cell extracts resolved by SDS-PAGE under nonreducing conditions. Disulfide linkage of GHRs reflects attainment of the active signaling conformation. Likewise, GH and GH-GH, but not G120R, caused Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) activation. Notably, G120R-G120R, despite its lack of an intact site 2 in either dimer partner, also promoted GHR disulfide linkage and JAK2 and STAT5 activation, albeit less potently than either GH or GH-GH. Time-course responses of the three agonists were similar in terms of JAK2 and STAT5 activation. Pretreatment of cells with our conformation-sensitive inhibitory monoclonal antibody, anti-GHR ext-mAb, prevented ligand-induced receptor activation for all three agonists. GHR was also rendered less immunoprecipitable by anti-GHR ext-mAb after treatment with these agonists. These results are important in that they indicate that a ligand with two intact binding sites 1 causes GHR to adopt similar conformational changes as does GH and thus triggers activation of JAK2 and downstream signaling. Furthermore, we infer that there is substantial flexibility in the GHR extracellular domain, such that it productively accommodates GH dimers that are much larger than GH.

Additive effects of a prolactin receptor antagonist, G129R, and herceptin on inhibition of HER2-overexpressing breast cancer cells.

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

Two wrongs can make a right: dimers of prolactin and growth hormone receptor antagonists behave as agonists.

Prolactin (PRL) and GH have two distinct binding sites (site 1 with high affinity; site 2 with low affinity) that each interact with a PRL receptor (PRLR) to form a functional receptor dimer that activates signal transduction. The G129R mutation in PRL and the G120R mutation in GH disrupt the structural integrity of site 2 such that the ligands retain the ability to bind to the first receptor with high affinity, but act as receptor antagonists. In this study, we examined the ability of monomeric and dimeric forms of these ligands, human (h) PRL and hGH, and their antagonists (hPRL-G129R and hGH-G120R) to 1) bind to PRLRs; 2) induce conformational changes in PRLRs; 3) activate signaling pathways associated with the PRLR; and 4) mediate cell proliferation in vitro. In contrast to monomeric hPRL-G129R, homodimeric hPRL-G129R induced PRLR dimerization; activated Janus family of tyrosine kinases 2/signal transducer and activator of transcription 5, Ras/Raf/MAPK kinase/Erk, and phosphatidylinositol 3-kinase/Akt signaling; and stimulated Nb2 cell proliferation. Similarly, homodimeric hGH-G120R was able to mediate signaling via the PRLR and to stimulate Nb2 cell proliferation. These experiments demonstrate that a ligand must have two functional binding sites, but that these may be site 1 plus site 2 or two site 1's, to elicit receptor-mediated signal transduction. The size of the ligand plays less of a role in receptor activation, suggesting that the extracellular portion of the PRLR (and possibly the GH receptor) is rather flexible and can accommodate larger ligands. These findings may have implications for designing multifunctional therapeutics that target this class of cytokine receptors.

The role of human prolactin and its antagonist, G129R, in mammary gland development and DMBA-initiated tumorigenesis in transgenic mice.

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Functional and structural outcomes following lensectomy for ectopia lentis.

PURPOSE: High refractive errors and optical aberrations reduce vision when the lens edge bisects the pupil. We studied outcomes of eyes with ectopia lentis following lensectomy. METHODS: Charts of 11 consecutive patients with bilateral ectopia lentis who underwent lensectomy-anterior vitrectomy in at least one eye from 1985 to 2004 were reviewed. Eighteen eyes were operated. One eye was excluded due to short-term follow-up (<2 years). RESULTS: Mean age at surgery was 7.7 years (2 to 17 years). Median follow-up after lensectomy was 10 years (range 2 to 16 years). Six eyes were followed for 6 to 10 years, and another six eyes were followed for 11 to 16 years. Patient diagnoses included Marfan syndrome (nine eyes), ectopia lentis et pupillae (three eyes), simple ectopia lentis (two eyes), homocystinuria (two eyes), and sporadic spherophakia (one eye). Preoperative best-corrected visual acuity (BCVA) ranged from 20/60 to light perception, and postoperative BCVA ranged from 20/20 to 20/100 (14 eyes were at least 20/30). Complications included posterior vitreous detachment (two eyes, 12%), glaucoma (one eye, 6%), transient ocular hypertension (one eye, 6%), wound dehiscence with iris incarceration (one eye, 6%), transient vitreous hemorrhage (one eye, 6%), and peripheral anterior synechiae (one eye, 6%). No retina detached. CONCLUSIONS: Our cohort of patients with long-term follow-up shows that pars plana lensectomy can be successful in restoring vision when conservative measures fail.

Development of a prolactin receptor-targeting fusion toxin using a prolactin antagonist and a recombinant form of Pseudomonas exotoxin A.

Human prolactin (hPRL) promotes the proliferation and differentiation of mammary epithelial cells during mammary gland development and has been linked to breast tumor development. The receptor for hPRL (hPRL-R) is elevated in a majority of human breast tumors, suggesting the overexpression of hPRL-R makes cancer cells highly sensitive to the mitogenic and anti-apoptotic activity of hPRL. These findings provide the rationale for the development of hPRL-R targeting breast cancer therapeutics. Previously, an hPRL-R antagonist, G129R, was developed that competitively binds to the hPRL-R resulting in growth inhibition and the induction of apoptosis in certain types of breast cancer cells. To further increase the potency of G129R, we fused G129R to a truncated form of Pseudomonas exotoxin A (PE(40)) that lacks the cell recognition domain of the toxin but retains the domains necessary for PE(40)_ to translocate into the cytosol and inhibit protein synthesis. We postulated that the fusion of G129R with PE(40)-KDEL would (1) deliver the recombinant toxin to breast cancer cells where hPRL-R is overexpressed; (2) block hPRL signaling via its G129R moiety; and (3) inhibit protein synthesis via its PE(40)-KDEL moiety. We demonstrate that the fusion toxin can competitively bind to hPRL-Rs on T-47D human breast cancer cells and inhibit STAT5 phosphorylation induced by hPRL. In addition, we show that G129R-PE(40)-KDEL is selectively cytotoxic to breast cancer cell lines expressing the hPRL-R and that cell death is associated with the inhibition of protein synthesis and does not involve caspase mediated apoptosis.

Unmodified prolactin (PRL) and S179D PRL-initiated bioluminescence resonance energy transfer between homo- and hetero-pairs of long and short human PRL receptors in living human cells.

We have used bioluminescence resonance energy transfer (BRET) to examine the interaction between human prolactins (PRLs) and the long (LF) and two short isoforms (SF1a and SF1b) of the human PRL receptor in living cells. cDNA sequences encoding the LF, SF1a, and SF1b were subcloned into codon-humanized vectors containing cDNAs for either Renilla reniformis luciferase (Rluc) or a green fluorescent protein (GFP(2)) with a 12- or 13-amino acid linker connecting the parts of the fusion proteins. Transfection into human embryonic kidney 293 cells demonstrated maintained function of Rluc and GFP(2) when linked to the receptors, and confocal microscopy demonstrated the localization of tagged receptors in the plasma membrane by 48 h after transfection. All three tagged receptors transduced a signal, with the LF and SF1a stimulating, and SF1b inhibiting, promoter activity of an approximately 2.4-kb beta-casein-luc construct. Both unmodified PRL (U-PRL) and the molecular mimic of phosphorylated PRL, S179D PRL, induced BRET with all combinations of long and short receptor isoforms except SF1a plus SF1b. No BRET was observed with the site two-inactive mutant, G129R PRL. This is the first demonstration, 1) that species homologous PRL promotes both homo- and hetero-interaction of most long and short PRLR pairs in living cells, 2) that both U-PRL and S179D PRL are active in this regard, and 3) that there is some aspect of SF1a-SF1b structure that prevents this particular hetero-receptor pairing. In addition, we conclude that preferential pairing of different receptor isoforms is not the explanation for the different signaling initiated by U-PRL and S179D PRL.

In vitro studies of baicalin alone or in combination with Salvia miltiorrhiza extract as a potential anti-cancer agent.

Traditional Chinese herbal medicines (TCM) that for centuries have been used in disease prevention and treatment are finding use as alternatives to Western cancer therapies. From a panel of TCM, we chose four compounds representing two functional classes of botanicals, the purified plant flavins scutellarin (a circulatory stimulant) and baicalin (antipyretic), and two extracts purified from Salvia miltiorrhiza (SM-470, circulatory stimulant) and Camellia sinensis (Cam-300, antipyretic), and examined their anti-proliferation effects on the human breast cancer cell lines MCF-7 and T-47D. All four compounds inhibited MCF-7 and T-47D cell proliferation, baicalin being the most potent inhibitor. Moreover, the combination of compounds from different classes offers enhanced potential therapeutic benefits; the combination of SM-470 with scutellarin, Cam-300 or baicalin, augmented the inhibition of cell proliferation. A synergistic inhibitory effect on MCF-7 cell proliferation was also observed when SM-470 and baicalin were applied together. In contrast, inhibition of T-47D cell proliferation using the same combination was dependent on baicalin only. The anti-proliferative effects of these compounds can be extended to other cancer types; the human head and neck cancer epithelial cell lines CAL-27 and FaDu were also sensitive to the four drugs. Overall, SM-470, Cam-300, scutellarin and baicalin inhibited the proliferation of human breast cancer cells and CAL-27 and FaDu cells with different potency. Baicalin and SM-470 in combination produced additive effects, suggesting these compounds may function by different mechanisms. T-47D, MCF-7, and FaDu cells may be useful in exploring the cellular and molecular mechanisms of action of baicalin and SM-470.

Ophthalmic manifestations of Wolf-Hirschhorn syndrome.

PURPOSE: Wolf-Hirschhorn syndrome is caused by partial deletion of the short arm of chromosome 4 (4p-). Common features include developmental delay, microcephaly, seizures, craniofacial anomalies, mental retardation, and cardiac defects. This article further describes the ocular manifestations of this rare disorder. METHODS: Charts of patients with 4p- from the University of Arkansas (n = 3) and the University of Minnesota (n = 7) were reviewed. Diagnosis was made by a geneticist and was confirmed by karyotype. Cytogenetic reports were available for review in eight patients. RESULTS: Ten patients (six females and four males) aged 4 months to 11 years were included. Ophthalmic findings included exodeviation (9/10), nasolacrimal obstruction (6/10), shallow orbits (3/10), epicanthal folds (3/10), foveal hypoplasia (3/10), upper lid coloboma (2/10), optic disk anomalies (2/10), downslanting palpebral fissures (2/10), microcornea (2/10), hypertelorism (1/10), nystagmus (1/10), and chorioretinal coloboma (1/10). Eight patients with 4p- had break points ranging from band 4p14 to 4p16.3. CONCLUSIONS: This study expands on previous reports of the ophthalmic phenotype in 4p- and includes the additional findings of foveal hypoplasia, nystagmus, shallow orbits, epicanthal folds, and upper lid colobomas. Ophthalmic findings in 4p- are variable, likely related to the size of the deletion.