Deletion of the immunoglobulin domain of IL1RAPL1 results in nonsyndromic X-linked intellectual disability associated with behavioral problems and mild dysmorphism.

X-Linked intellectual disability accounts for a significant fraction of males with cognitive impairment. Many of these males present with a non-syndromic phenotype and presently mutations in 17 X-linked genes are associated with these patients. Mutations in IL1RAPL1 have been found in multiple families with non-syndromic X-linked intellectual disability. All of the published mutations predict loss of function of the protein. We have identified an additional two families with deletions of a portion of the gene that give rise to cognitive impairment, as well as some behavioral problems and mild dysmorphism. Our clinical findings better delineate the phenotypic spectrum associated with IL1RAPL1 mutations.

A role of the (pro)renin receptor in neuronal cell differentiation.

The (pro)renin receptor [(P)RR] plays a pivotal role in the renin-angiotensin system. Experimental models emphasize the role of (P)RR in organ damage associated with hypertension and diabetes. However, a mutation of the (P)RR gene, resulting in frame deletion of exon 4 [Delta4-(P)RR] is associated with X-linked mental retardation (XLMR) and epilepsy pointing to a novel role of (P)RR in brain development and cognitive function. We have studied (P)RR expression in mouse brain, as well as the effect of transfection of Delta4-(P)RR on neuronal differentiation of rat neuroendocrine PC-12 cells induced by nerve growth factor (NGF). In situ hybridization showed a wide distribution of (P)RR, including in key regions involved in the regulation of blood pressure and body fluid homeostasis. In mouse neurons, the receptor is on the plasma membrane and in synaptic vesicles, and stimulation by renin provokes ERK1/2 phosphorylation. In PC-12 cells, (P)RR localized mainly in the Golgi and in endoplasmic reticulum and redistributed to neurite projections during NGF-induced differentiation. In contrast, Delta4-(P)RR remained cytosolic and inhibited NGF-induced neuronal differentiation and ERK1/2 activation. Cotransfection of PC-12 cells with (P)RR and Delta4-(P)RR cDNA resulted in altered localization of (P)RR and inhibited (P)RR redistribution to neurite projections upon NGF stimulation. Furthermore, (P)RR dimerized with itself and with Delta4-(P)RR, suggesting that the XLMR and epilepsy phenotype resulted from a dominant-negative effect of Delta4-(P)RR, which coexists with normal transcript in affected males. In conclusion, our results show that (P)RR is expressed in mouse brain and suggest that the XLMR and epilepsy phenotype might result from a dominant-negative effect of the Delta4-(P)RR protein.

Prolactin antagonist-endostatin fusion protein as a targeted dual-functional therapeutic agent for breast cancer.

In previous studies (Chen, W. Y. et al., Clin. Cancer Res., 5:3583-3593, 1999; Chen, N Y. et al., Int. J. Oncol., 20:813-818, 2002), we have demonstrated the ability of the human prolactin (hPRL) antagonist, G129R, to inhibit human breast cancer cell proliferation in vitro and to slow the growth rate of tumors in mice. We further revealed that the possible mechanisms of G129R antitumor effects act through the induction of apoptosis via the regulation of bcl-2 gene expression. It has been established that to sustain tumor growth, it is necessary for the development of a network of blood vessels to bring in nutrients, a process called angiogenesis. The disruption of angiogenesis has been proven to be an effective strategy to cause regression of certain tumors. One of the best-studied angiogenesis inhibitors is endostatin, which acts through the inhibition of endothelial cells. In this study, we combine the anti-breast tumor effects of G129R and the antiangiogenic effects of endostatin by creating a novel fusion protein (G129R-endostatin) specifically for breast cancer therapy. The data presented here demonstrated that this novel fusion protein was able to bind to the PRL receptor (PRLR) on T-47D human breast cancer cells and inhibit the signal transduction induced by PRL. At the same time, G129R-endostatin inhibited human umbilical vein endothelial cell (HUVEC) proliferation and disrupted the formation of endothelial tube structures with potency similar to that of endostatin. More importantly, the therapeutic efficacy of G129R-endostatin was confirmed using a mouse breast cancer cell line 4T1 in vivo. G129R-endostatin has a significantly prolonged serum half-life as compared with that of G129R or endostatin alone, and exhibited greater tumor inhibitory effects than G129R and endostatin individually or in combination. Taken together, these data demonstrate the dual therapeutic effects of G129R-endostatin, and suggests that this fusion protein has great promise as a novel anti-breast cancer agent.

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.

Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv).

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.