Phospholipid transfer protein (PLTP) and cholesterol metabolism

Abstract

A method for controlling cholesterol metabolism in a host, a screening assay for agents that can control cholesterol metabolism, and the agents that may be identified are practiced and determined in relation to the expression of PLTP in HepG2 cells and a clone from these cells, designated HepG2/PLTPpLuc. Particular agents that are covered comprise camptothecin, topotecan, derivatives thereof, metabolic byproducts thereof and small molecule mimics thereof.

1 - 20 . (canceled)  
     
     
         21 . A reporter construct for use in a high throughput process of screening for small molecules that regulate phospholipids transfer protein gene promoter activity comprising: 
 a. a phospholipids transfer protein gene promoter associated with a reporter gene; and    b. a resistance marker gene for selection and identification of clones containing the reporter construct.    
     
     
         22 . A cell line containing the reporter construct of  claim 21 .  
     
     
         23 . A cell line of  claim 22 , wherein said reporter gene is a luciferase reporter gene.  
     
     
         24 . A cell line of  claim 22  wherein said resistance marker gene is neomycin.  
     
     
         25 . The cell line of  claim 24 , wherein said resistance marker gene is selectable using an aminoglycoside antibiotic.  
     
     
         26 . The cell line of  claim 25 , wherein said aminoglycoside antibiotic is G418.  
     
     
         27 . The cell line of  claim 22 , selected from the group consisting of HepG2, HepG2/PLTP p Luc and clones thereof.  
     
     
         28 - 29 . (canceled)  
     
     
         30 . A process for monitoring the pharmacological regulation of phospholipid transfer protein gene promoter activity comprising measuring the expression of a reporter gene, said reporter gene expressed by the cell line of  claim 22 .  
     
     
         31 . A high throughput process of screening for small molecules that modulate phospholipids transfer protein gene promoter activity comprising: 
 a. isolating a biological sample;    b. introducing a reporter construct comprising a phospholipids transfer protein gene promoter associated with a reporter gene, and a resistance marker gene for selection and identification of clones containing the reporter construct into said biological sample;    c. contacting said biological sample with test agents for modulating activity of said reporter construct; and    d. measuring said biological sample for expression of said gene construct.    
     
     
         32 . A high throughput process of screening for small molecules that regulate phospholipids transfer protein gene promoter activity comprising: 
 a. culturing the cells from the cell line of  claim 22  for a period of time sufficient to induce the phospholipids transfer protein gene promoter activity with one of the following components: 
 i. said small molecule being tested for its ability to regulate phospholipids transfer protein gene promoter activity; or  
 ii. a compound known to regulate phospholipids transfer protein gene promoter activity to act as a positive control; or  
 iii. cell culture media without any small molecules or compounds to act as a negative control;  
   b. measuring luciferase activity in said cells;    c. comparing luciferase activity in the cells containing said small molecules with cells containing a compound known to induce phospholipids transfer protein gene promoter activity;    d. comparing luciferase activity in the cells containing said small molecules with cells containing no small molecules or compounds; and    e. establishing the amount of phospholipids transfer protein gene promoter activity using a standard curve for luciferase activity.    
     
     
         33 - 39 . (canceled)  
     
     
         40 . A method for estimating the course and extent of diseases, selected from the group consisting of Tangier's disease, familial HDL deficiency disease, and other diseases associated with deficiencies in uptake and removal of lipid molecules in a mammal comprising measuring the presence and amount of phospholipids transfer protein in said mammal.  
     
     
         41 . A method for assessing the lipid metabolic state in a mammal comprising measuring the presence and amount of phospholipids transfer protein in said mammal.  
     
     
         42 . The method of  claim 40 , comprising the steps of: 
 a. preparing at least one biological sample taken from a mammal in which said phospholipids transfer proteins are suspected to be present;    b. immobilizing a material selected from the group consisting of the biological sample of Step (a) and a binding partner to phospholipids transfer proteins and phospholipids transfer protein related material, on a suitable substrate, and labeling one of the materials not mobilized;    c. incubating said sample with said binding partner for a period of time sufficient for said binding partner to become bound to any phospholipids transfer proteins and phospholipids transfer protein related material present in said sample;    d. removing any unbound material from step (c); and    e. comparing the amount of label bound to said sample to a standard.    
     
     
         43 . The method of  claim 42 , wherein said binding partner is selected from the group consisting of a receptor for a phospholipids transfer protein, and an antibody reactive with or capable of binding to said phospholipids transfer protein.  
     
     
         44 . The method of  claim 43 , wherein said antibody to said phospholipids transfer protein is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, synthetic antibodies, bispecific antibodies, humanized antibodies, and chimeric antibodies.  
     
     
         45 . The method of  claim 40  comprising a method for monitoring the level of high or low density lipoprotein in said mammal.  
     
     
         46 . The method of  claim 40 , comprising a method for diagnosing the likelihood or onset, and/or for monitoring the course or severity of a pathology selected from the group consisting of Tangier's disease, familial HDL deficiency disease, atherosclerosis, vascular disease, and hypercholesterolemia.  
     
     
         47 - 49 . (canceled)  
     
     
         50 . The method of  claim 31  or  32  for identifying small molecules that regulate PLTP gene expression or protein levels in a mammal.  
     
     
         51 . The method of  claim 31  or  32  for identifying small molecules that regulate pre-HDL levels in a mammal.