Patent Application: US-41859499-A

Abstract:
a method for using nordihydroguiaretic acid derivatives , in particular tetramethyl nordihydroguiaretic acid and tetraglycinal nordihydroguiaretic acid , for the localized treatment of tumors induced by human papillomavirus .

Description:
ndga derivatives were sythesized chemically ( 5 ). cell line c3 is a hpv16e + l plus activated ras transformed cell line of c57 bl / 6kh origin provided by w . martin kast of loyola university medical center , chicago , ill ., u . s . a . it is maintained and cultivated as described by greenstone et al ( 11 ) and feltkamp et al . ( 12 , 13 ). standard procedure for the preparation of meso - 1 , 4 - bis [ 3 , 4 -( dimethylaminoacetoxy ) phenyl ]-( 2r , 3s )- dimethylbutane hydrochloride salt tetraglycinyl ndga , g 4 n . to a dichloromethane ( 250 ml ) solution containing ndga ( 12 . 8 g , 42 . 3 mmol , 1 . 0 equiv ) and n , n ,- dimethylglycine ( 26 . 2 g , 254 mmol , 6 . 0 equiv ) were added dcc ( 52 . 4 g , 254 mmol , 6 . 0 equiv ) and dmap ( 2 . 32 g , 18 . 9 mmol , 1 . 0 equiv ). the reaction mixture was stirred for 24 h under nitrogen at room temperature . after the reaction mixture was filtered , the solution was concentrated under reduced pressure . acetone ( 250 ml ) was then added into the reaction flask and the solution was bubbled with excess hcl ( g ). the water - soluble precipitate was dissolved in h 2 o and re - precipitate twice at room temperature from acetone to give ( 1 ) ( 29 , 2 g , 36 . 8 mmol ) as a white solid in 87 % yield . proton nmr spectra were obtained on a varian unity - 400 ( 400 mhz ) spectrometer by use of d 2 o solvent and tsp as internal standard . carbon - 13 nmr spectra were obtained on a varian unity - 400 ( 400 mhz ) spectometer by used of d 2 o as solvent . carbon - 13 chemical shifts are referenced to the tsp singlet ( δ0 . 0 ppm ). general procedure . all reactions were carried out in oven - dried glassware ( 120 ° c .) under an atmosphere of nitrogen , unless as indicated otherwise . acetone , dichloromethane , 1 , 4 - dioxane , ethyl acetate , hexanes , and tetrahydrofuran were purchased from mallinckrodt chemical co . acetone was dried with 4a molecular sieves and distilled . dichloromethane , ethyl acetate , and hexanes were dried and distilled from cah 2 . 1 , 4 - dioxane and tetrahydrofuran were dried by distillation from sodium and benzophenone under an atmosphere of nitrogen . nordihydroguaiaretic acid was purchased from fluka chemical co . n , n ′- dicyclohexylcarbodiimide ( dcc ), 4 - dimethylaminopyridine ( dmap ), morpholine , triethylamine , and potassium carbonate were purchased from merck inc . 1bromo - 3 - chloropropane , n , n - dimethylglycine , and methyl phosphorodichloridate were purchased from aldrich chemical co . analytical thin layer chromatography ( tlc ) was performed on precoated plates ( silica gel 60 f - 254 ), purchased from merck inc . gas chromatographic analyses were performed on a hewlett - packard 5890 series ii instrument equipped with a 25 - m cross - linked methyl silicone gum capillary column ( 0 . 32 mm i . d .). nitrogen gas was used as a carrier gas and the flow rate was kept constant at 14 . 0 ml / min . the retention time tr was measured under the following conditions : injector temperature 260 ° c ., isothermal column temperature 280 ° c . gas chromatography and low resolution mass spectral analyses were performed on a hewlett - packard 5890 series ii instrument equipped with a hewlett - packard 5971a mass selective detector and a capillary hp - 1 column . separations by medium - pressure liquid chromatography ( mplc ) were performed at a flow rate of 120 ml / h by use of a jasco model 880 - pu intelligent hplc pump . the mplc packing material , reversed phase silica gel c18 ( particle size 0 . 035 - 0 . 070 mm ), was purchased from knauer co . purification by gravity column chromatography was carried out by use of merek reagents silica gel 60 ( particle size 0 . 063 - 0 . 200 mm , 70 - 230 mesh astm ). infrared ( jr ) spectra were measured on a bomem michelson series ft - ir spectrometer . the wave numbers reported are referenced to the polystyrene 1601 cm ˜ 1 absorption . absorption intensities are recorded by the following abbreviations : s , strong ; m , medium ; w , weak . proton nmr spectra were obtained on a varian unity - 400 ( 400 mhz ) spectrometer by use of d2o as solvent and 3 -( trimethylsilyl ) propionic acid , sodium salt as internal standard . carbon - 13 nmr spectra were obtained on a varian unity - 400 ( 100 mhz ) spectrometer by used of d2o as solvent . carbon - 13 chemical shifts are referenced to the center of the 3 -( trimethylsilyl ) propionic acid , sodium salt singlet ( 6 0 . 0 ppm ). multiplicities are recorded by the following abbreviations : s , singlet ; d , doublet ; t , triplet ; q , quartet ; m , multiplet ; j . coupling constant ( hertz ). high - resolution mass spectra were obtained by means of a jeol jms - hx110 mass spectrometer . meso ˜ 1 , 4bis [ 3 , 4 ( dimethyleminoacetoxy ) phenyl ]- 2 , 3 - dilnethylbutane hydrochloride salt ( 2 ). to a solution of ndga ( 1 , 12 . 81 g , 42 . 37 mmol , 1 . 0 equiv ) and n , n - dimethylglycine ( 26 . 21 g , 254 . 2 mmol , 6 . 0 equiv ) in dichloromethane ( 250 ml ) was added dcc ( 52 . 45 g , 254 . 2 mmol , 6 . 0 equiv ) and dmap ( 5 . 176 g , 42 . 37 mmol , 1 . 0 equiv ). the reaction mixture was stirred for 24 h under nitrogen at room temperature . after dicyclohexylurea in the reaction mixture was filtered off , the resultant solution was concentrated under reduced pressure . acetone ( 250 ml ) was then added into the residue and the resultant solution was bubbled with excess hcl ( g ). the precipitate was dissolved in water and re - precipitated twice by use of acetone at room temperature to give 2 ( 28 . 97 g , 36 . 86 mmol ) as a white solid in 87 % yield : 1h nmr ( d2o , 400 mhz ) o 0 . 78 ( d , j = 6 . 0 hz , 6 h . 2 × ch3 ), 1 . 73 ( m , 2 h . 2 × ch ), 2 . 38 ( dd , j = 13 . 2 , 9 . 6 hz , 2 h . 2 × arch ), 2 . 78 ( dd , j = 13 . 2 , 4 . 4 hz , 2 h . 2 × arch ), 3 . 03 ( s , 24 h . 8 × ch3n ), 4 . 53 ( s , 8 h . 4 × ch2n ), 7 . 22 ( m , 4 h . 4 × arh ), 7 . 29 ( d , j = 8 . 4 hz , 2 h . 2 × arh ); 13c nmr ( d2o , 100 mhz ) 0 18 . 11 , 40 . 82 , 41 . 73 , 46 . 75 , 59 . 59 , 125 . 79 , 126 . 58 , 131 . 63 , 140 . 66 , 142 . 47 , 146 . 11 , 167 . 84 ; ir ( kbr ) 3461 ( br ), 2963 ( m ), 1777 ( s , c ═ o ), 1620 ( m ), 1478 ( m ), 1377 ( m ), 1210 ( m ), 1106 ( m ), 961 ( w ), 852 ( w ) cm - l ; ms ( fab ) of ( 2 - 4 hcl ) m / z ( relative intensity ) 643 ( m +, 30 ), 600 ( 20 ), 558 ( 43 ), 515 ( 20 ), 473 ( 42 ), 430 ( 13 ), 388 ( 26 ), 185 ( 18 ), 93 ( 38 ), 58 ( 100 ), 44 ( 22 ); hrms ( fab ) of ( 2 - 4 hcl ) calcd for c34hson4og 642 . 3628 , found 642 . 3614 ; anal . calcd for c34hs4n4ogcl4 : c , 51 . 78 ; h . 6 . 90 ; n . 7 . 10 ; o . 16 . 23 . found : c , 51 . 70 ; h . 6 . 85 ; n . 7 . 05 ; o . 16 . 21 . effect of m 4 n and several other ndga derivatives of sp1 - regulated hpv e 6 / e 7 promoter activity the effect of m 4 n and several other ndga derivatives of sp1 - regulated hpv e 6 / e 7 promoter activity was examined using luciferase as a reporter . the assay depends upon dna transfection of the hpv16 lcr ( p 97 promoter ) fused to the luciferase reporter gene into c33a cells by calcium phosphate methods . c33a is a cervical tumor cell line ( atcc accession no . htb - 31 ) that does not contain any integrated hpv dna , but has transcription factors necessary for a robust expression of the hpv early gene promoter . one day following dna transfection various drug concentrations dissolved with the help of dimethyl sulfoxide ( dmso ) were added to the cells . thirty hours after drug treatment ( so that the assay is complete within the standard forty - eight hours for transient transfection experiments ), the cells were lysed and specific luciferase activity was determined ( luciferase assay systems , promega , u . s . pat . no . 5 , 283 , 179 ). as the m 4 n drug concentration was increased the specific luciferase activity decreased . the results ( shown in fig2 ) demonstrate that m 4 n dramatically reduces sp1 regulated transcription initiation at the hpv e 6 / e 7 promoter in luciferase assay . inhibition of e 6 / e 7 mrna synthesis following m 4 n treatment inhibition of e 6 / e 7 mrna synthesis following m 4 n treatment was measured by rt - pcr in cervical cell line c 3 . relative rt - pcr was performed with quantities of total cellular rna standardized to the cell numbers counted . the rt - pcr product was analyzed on a 2 % agarose gel . the results are shown in fig3 . the rt - pcr results indicated that the amplified cdnas of the expected size for e7 ( 321 bp ) and e6 ( 204 bp ) were detected in the dmso treated cells as early as cycle 22 of amplification . these same products were barely detectable in the drug treated rna extracts following 30 cycles of amplification . no amplified products were detected for the no template pcr control or from total rna extracts of the hpv16 - negative c33a cell line . inhibition of cervical c3 cell growth by m 4 n treatment hpv - 16 transformed immortal mouse epithelial cells ( c3 cells ) were plated at a density of 10 5 cells per vial . after 24 hours , ½ of the vials were given growth media containing 40 μmm 4 n dissolved in 1 % dmso while the other half were given growth media containing only 1 % dmso . the results are shown in fig4 a . within 24 hrs a difference in cell morphology between drug treated and control c3 cells was observed . the growth and division of the drug treated cells was markedly reduced in comparison to the untreated control , while the fraction of viable cells compared to the total cell count remained constant for both drug treated and dmso only control cells . this indicates that m 4 n dramatically reduces cell division . the effect on c3 growth following removal of m 4 n from the medium was also examined . c3 cells were plated at a density of 10 4 cells per vial . at time = 0 , ⅔ of the vials were given growth media supplemented with 40 μm m 4 n in 1 % dmso . the remaining vials were given growth media containing only 1 % dmso . after 73 hours , ½ of the vials that had received m 4 n in their growth media were washed and media containing only 1 % dmso was added . the other ⅔ of the cell vials were washed and replaced with the same media administered before . the results , shown in fig4 b , indicate that the rate of cell growth was not notably increased in m 4 n treated sample following the change to drug - free media , indicating that m 4 n continues to significantly reduce cell division even after its removal from the extracellular environment . analysis of cellular gene expression in c3 cells before and after 72 hrs of drug treatment gene expression with 9600 gene arrays was studied ( fig5 ). five micrograms each of poly a + rna from 72 hrs . m 4 n ( 40 μm ) treated ( c 3 m 4 n ) and non - treated ( c 3 dmso ) was used in a pair of human 9600 gene array hybridization study according to the procedure described in genomics 51 , 313 - 324 1998 . the hybridization image was captured by a color video camera with a nikon 55 mm af micro niko lens and digitized by a macintosh lc630 computer . such detection via enzyme substrate reaction of color - forming enzymes in either single or dual - color mode is reproducible and extremely sensitive ( can detect & lt ; 5 copies of transcript per cell with rnas from 10 7 cells ). the computer print outs showing differentially expressed genes ( c 3 m 4 n / c 3 dmso & gt ; 10 and c 3 dmso / c 3 m 4 n & gt ; 10 ) were listed for examination . image files in tiff format and data files in ms excel format are kept on zip diskette . gene names and clone id numbers are available for obtaining image clones for future northern blot confirmation . among a group of genes that are either up - regulated or down - regulated 72 hrs after m 4 n treatment , the following are those specifically related to cell division and apoptosis . the following cell cycle related genes are greatly unregulated in response to m 4 n : the following cell cycle related genes are greatly downregulated in response to m 4 n : at earlier time points , such as after one hour drug treatment , e 6 / e 7 level was found to be similar with those in control cells while after 4 . 5 hrs , e 6 / e 7 were no longer detectable by rt - pcr ( 39 ). gene expressions with 9600 gene arrays can be repeated with rna isolated from these short - time treated cells ( 1 hour and 5 hours ) in order to further pin down the initial cellular effects of the drug . targeting c3 tumor growth in mice by local injection of m 4 n thirty six c57bl - 16 ncr mice were injected with 5 × 10 5 c3 cells between the shoulders on the backs of the mice . twenty four of the mice developed tumors within 20 days . they were used for the first set of experiments . daily injection ( 50 μl - 100 μl of m 4 n or m 4 n / g 4 n ) ( 200 mg / ml m 4 n in dmso , 200 mg / ml g 4 n in pbs ) showed profound effect in tumor growth in animals , as shown in tables 1 and 2 , fig6 and 7 . *** lesion contained mostly necrotic cells as also found in lesions from mouse 6 , 7 , 11 , 14 , 15 , 17 , 19 , 21 , 28 , 19 ( fig6 ). there were no lesions left in mouse # 11 and # 22 following drug treatments . tumors found in control mouse # 1 , 2 , 3 , 4 contained growing cells ( fig2 ). 36 c57bl - 16ncr mice were injected with 5 × 10 5 c3 cells / mouse . injections were 100 μl made subcutaneously between the shoulders on the backs of the mice . cells were suspended in low - salt hbss and suspension uniformity was maintained by gentle vortexing . 24 mice developed tumors . their lesion sizes were measured by dial caliper . these mice were shaved , weight and treatment begun ( day 1 ). four mice were sequestered as controls . control mice received 50 μl dmso injected intratumorally daily . experimental mice ( 10 ) received 50 μl m 4 n dissolved in dmso ( 200 mg / ml ). an additional 10 mice received m 4 n treatments for 8 days followed by g 4 n treatments ( 50 μl , 200 mg / ml in pbs ) daily for 8 days . injections were made to several regions of tumor . mice anesthetized with ether or metaphane prior to injection . c57bl - 16ncr female mice from ncl were used in this experiment . tetraglycinal ndga ( g 4 n ) was freshly made everyday in pbs in concentrations of 75 mg / ml . injections of 0 . 05 ml for group 1 , 0 . 1 ml for groups 2 and 4 , and 0 . 2 ml for group 3 per treatment were made for a period of 6 days . experiments lasted seven days . body weights were determined before and after six days of injection . no significant weight changes were observed during the experimental period . all treated mice , controls ( mouse numbers 1 - 4 ) and experimental mice ( mouse numbers 6 , 7 , 9 , 10 , 11 , 12 , 14 , 15 , 16 , 17 m 4 n numbers 18 - 22 , 24 , 26 - 29 m 4 n / g 4 n ) exhibited swelling . measurements of lesion sizes were made by dial caliper . some mice experienced mild bleeding due to injection . the treatment regimen and results were as follows : day 10 : mice weighed again . all mice exhibited growth up to two grams . day 13 : all mice have raised skin but to very different degrees . the skin of one m 4 n treated mouse (# 7 ) has split open through which the “ dried - out tumor ” fell out . day 15 : one m 4 n treated mouse (# 17 ) died due to overdose of anesthesia / handling . the skin at the lesion site of # 17 cracked with the “ dried - out tumor ” showing . it was dissected , and lesion excised and weighed . day 16 : four more m 4 n treated mice (# 6 , 14 , 15 , 16 ), three m 4 n / g 4 n treated mice (# 19 , 21 , 28 ) and one control mouse (# 2 ) were euthanized , dissected and weighed . remaining control mice (# 1 , 3 , 4 ) were examined non - invasively and were carrying tumors . day 21 : tumor sizes from control mice were measured by dial caliper . observation : the skin at the lesion sites of mouse # 10 and # 12 ( m 4 n treated regions ) cracked with the “ dried - out tumor ” showing . day 24 : mouse # 7 skin recovered completely . the experiment was terminated on this date . all remaining mice , m 4 n treated (# 7 , 9 , 10 , 11 , 12 ) and m 4 n / g 4 n treated (# 18 , 20 , 24 , 26 , 29 ) were euthanized , dissected , examined and weighed . the effects of m 4 n and m 4 n / g 4 n on c3 tumor growth in mice are summarized in tables 1 and 2 and fig5 and 6 . table 1 shows the drug effect on c3 cell growth in mice carrying single tumors . the average weight of four excised tumors of the control group was 1 . 48 g while weights of lesions from m 4 n treated and m 4 n / g 4 n treated were 0 . 142 and 0 . 51 g respectively . drug treated lesions consisted mainly of dried out necrotic cells ( fig6 ). tumors from the control group appeared homogenous and contained actively growing cells . table 2 shows the drug effect on c3 tumor growth in mice carrying multiple tumors . in this study , drug was injected into one of the tumors . the average weight of untreated tumors was 1 . 77 g while that of m 4 n treated lesions was 0 . 15 g . similar results were obtained following m 4 n / g 4 n injection — the average weight of untreated tumors was 1 . 27 g , while that of the drug treated lesions was only 0 . 103 g . the body weight changes of all mice during the entire experimental period appeared insignificant ( table 1 and 2 ). drug treated ( m 4 n ) and dmso vehicle - treated or untreated tumors ( con ) from two groups of mice were prepared for histopathology examination . the excised tumors were immediately fixed and then stored in 4 % formaldehyde in phosphate buffered saline . the fixed tissue was then dehydrated through a series of graded alcohols and xylene and embedded in paraffin . the paraffin tissue blocks were thin sectioned and stained for microscopy with hematoxylin and eosin . histopathology studies showed that the control tumors were unaffected by dmso treatment and continued to grow . they show the high nuclear / cytoplasmic ratio , pleomorphic nuclear changes , high mitotic figures , spindle like sarcoma shape , and infiltration into the surrounding tissue characteristic of cancer cells . in contrast , those tumors receiving m 4 n treatment discontinued growth shortly after treatment began . they demonstrate significant necrosis and are no longer viable . there is a small amount of drug precipitate visible at higher magnification , and focal areas show chronic inflammation and fibrosis . this healing effect leads to the shedding of these deceased tumor cells from the area . the same results are seen with m 4 n / g 4 n treatment as with the m 4 n treatment alone . however , since g 4 n is water - soluble , it can spread to a larger area of the tumor than m 4 n . g 4 n when used with m 4 n synergistically is more effective in trating tumors of larger than 2 cm 3 . effect of m 4 n on hsv - 1 skin infection in guinea pig the drug m 4 n was also tested in inhibition of hsv - 1 replication in skin infections in guinea pigs . guinea pig skin was pinched with needles and hsv - 1 suppression was applied topically to infect each pricked area . m 4 n was then applied to the pricked infected area following infection daily for 6 days . six areas of bared back skin of a guinea pig were punched sterilely with a 5 = din needle . two areas were infected with hsv - 1 ( hsv - c , culture supernatant , or isolated hsv in saline , hsv - sc ). the other four areas were infected with hsv - sc . fifteen minutes after infection , 30 μl of test compounds ( abds 1 , abds 2 , acv and m 4 n ( 4n ) in 60 mg / ml of dmso were applied to each punched infected region of an area , five times per day for six days . abds 1 and abds 2 were included as negative controls . the photograph in fig8 was taken at day 6 and shows the extent of hsv - 1 replication in the absence of drugs ( hsv - c , hsv - sc ), in the presence of ineffective drugs ( hsv - abds 1 , hsv - abds 2 ) and in the presence of effective drugs ( hsv - m 4 n and hsv - acv ). it can be seen that six large confluent blisters were developed in areas treated by hsv - c , hsv - sc , hsv - abds 1 , hsv - abds 2 , while no blisters were observed in infected areas following m 4 n ( 4n ) and acv treatments . clearcut results that m 4 n can block hsv replication were obtained in this model system as shown by the disappearance of the skin lesions and by no shedding of the virus 4 days after the drug treatment . initial animal studies also showed m 4 n to be non - toxic to mice at concentrations as high as 300 mg / kg when given intraperitoneally , and as high as 375 mg / kg when given either subcutaneously or by iv ( table 3 ) ( 6 ). administration of m 4 n directly into tumors as a drug delivery route provides several distinctive advantages . 1 ) m 4 n is a hydrophobic compound and is exceedingly soluble in dmso ( 200 mg / ml ). therefore only a small volume of the drug solution is needed for injection in order to achieve effective dosage of the drug . in the mouse study described in example 5 , above , daily injection of 50 μl to 100 μl for several days was sufficient to completely stop tumor growth in mice . there have been several previous studies on the use of large dosages ( 30 ml iv per treatment ) of dmso for treating diseases ( 14 ). the results were not conclusive ( 15 ). however , since tens of millions of people have been safely tested with large amounts of dmso worldwide in the past , it appears that dmso should be safe as a vehicle for drug delivery when only small volume of it will be used ( 16 ). 2 ) by injection in situ , a majority of the drug residue remains insoluble and concentrated in the tumor areas , and does not enter the circulatory system , thus whole body toxicity is avoided . in addition , since enough drug remains within the tumor to suppress its growth , continued injection of drug is unnecessary after relatively few treatments . in the mouse study of example 5 , tumor cells continued to die even after discontinuation of m 4 n injections . thus when drug is directly targeted , tumor size becomes the determining factor for the required amount of drug to be administered . the difference between whole body weights of a human vs . a mouse becomes irrelevant . in the mouse tumor studies , 20 mg / day for 10 days were more than sufficient to eliminate tumors . there should be no reason to use a higher dosage than this for treating a human tumor of comparable size ( 1 - 1 . 5 cm 3 ). this should reduce the risk considerably in human trials . references cited herein are listed below for convenience and are hereby incorporated by reference . 1 . baba , m . 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( 1995 ) ctl raised against a subdominant epitope offered as a synthetic peptide eradicate human papillonavirus type 16 - induced tumors . european journal of immunology 25 , 2638 - 2642 . 13 . feltkamp , m . c ., smits , h . l ., vierboom , m . p ., minaar , r . p ., b . m . drijfhout , j . w ., schegget , j ., melief , c . and kast , w . m . ( 1993 ) vaccination with cytotoxic t lymphocyte epitope - containing peptide protects against a tumor induced by human papillomavirus type 16 - transformed cells . eur . j immunol . 23 , 2242 - 2249 . 14 . jacob , s . w . and herschler ( 1986 ) pharmacology of dmso . academic press , inc . 15 . jack , c ., and torre , de la ( 1983 ) biological actions and medical applications of dimethyl sulfoxide . new york academy of sciences , new york , n . y . 16 . spruance , s . l ., mckeough . m . b . and cardinal , j . r . ( 1983 ) dimethyl sulfoxide as a vehicle for topical antiviral chemotherapy . ann . n . y . acad . sci . 411 , 28 - 33 .