Hedgehog signaling pathway function conserved in Tribolium segmentation In Drosophila , maintenance of parasegmental boundaries and formation of segmental grooves depend on interactions between segment polarity genes . Wingless and Engrailed appear to have similar roles in both short and long germ segmentation , but relatively little is known about the extent to which Hedgehog signaling is conserved . In a companion study to the Tribolium genome project , we analyzed the expression and function of hedgehog , smoothened , patched , and cubitus interruptus orthologs during segmentation in Tribolium . Their expression was largely conserved between Drosophila and Tribolium . Parental RNAi analysis of positive regulators of the pathway -LRB- Tc-hh , Tc-smo , or Tc-ci -RRB- resulted in small spherical cuticles with little or no evidence of segmental grooves . Segmental Engrailed expression in these embryos was initiated but not maintained . Wingless-independent Engrailed expression in the CNS was maintained and became highly compacted during germ band retraction , providing evidence that derivatives from every segment were present in these small spherical embryos . On the other hand , RNAi analysis of a negative regulator -LRB- Tc-ptc -RRB- resulted in embryos with ectopic segmental grooves visible during germband elongation but not discernible in the first instar larval cuticles . These transient grooves formed adjacent to Engrailed expressing cells that encircled wider than normal wg domains in the Tc-ptc RNAi embryos . These results suggest that the en -- wg -- hh gene circuit is functionally conserved in the maintenance of segmental boundaries during germ band retraction and groove formation in Tribolium and that the segment polarity genes form a robust genetic regulatory module in the segmentation of this short germ insect . Introduction The ontogenic stage at which the body plans of animals belonging to the same phylum reach maximum morphological similarity is called the phylotypic stage . In insects and other arthropods , the phylotypic stage is the elongated germband at which the three germ layers have formed and segments are morphologically evident along the entire anterior -- posterior axis -LRB- Sander 1997 -RRB- . In Drosophila , segment polarity genes , most of which are components of two major signal transduction pathways -LRB- the Wingless and Hedgehog signaling pathways -RRB- , control the formation of grooves between segments and anterior -- posterior patterning within each segment . Initially , pair-rule genes activate engrailed -LRB- en -RRB- expression at the anterior boundary of each parasegment and wingless -LRB- wg -RRB- at the posterior boundary DiNardo and O'Farrell -LRB- 1987 ; Howard et al. 1988 ; Ingham et al. 1988 -RRB- . Engrailed protein activates expression of hedgehog -LRB- hh -RRB- , which encodes a secreted protein that signals to surrounding cells -LRB- Hidalgo and Ingham 1990 ; Ingham and Hidalgo 1993 -RRB- . Hh signaling leads to the continued activation of wg , whose secreted protein product is necessary for the continued activation of en . This positive feedback loop ensures the interdependence of these three genes for the maintenance of each other 's expression until embryonic stage 9 -- 10 -LRB- Forbes et al. 1993 -RRB- . At the end of stage 10 , en expression becomes independent of wg , and it is around this stage that segmental boundaries are morphologically visible at the posterior edge of cells expressing en and hh . Evidence that this segment polarity network might be conserved is primarily based on the expression patterns of en , wg , and more recently , Hh pathway component genes in other insects as well as non-insect arthropods and annelids -LRB- Patel et al. 1989 ; Brown et al. 1994 ; Kraft and Jackle 1994 ; Nagy and Carroll 1994 ; Grbic et al. 1996 ; Peterson et al. 1998 ; Damen 2002 ; Dhawan and Gopinathan 2003 ; Simonnet et al. 2004 -RRB- . There is also limited data suggesting this network is functionally conserved among insects . Ectopic expression of Drosophila wg in Tribolium induces ectopic en in the anterior half of the parasegment suggesting functional conservation of the wg -- en interaction -LRB- Oppenheimer et al. 1999 -RRB- . wg and/or en have been implicated by RNAi analyses in the proper formation of segmental boundaries in the milkweed bug Oncopeltus faciatus -LRB- Angelini and Kaufman 2005 -RRB- , the honey bee Apis mellifera -LRB- Beye et al. 2002 -RRB- , the blowfly Lucilia sericata -LRB- Mellenthin et al. 2006 -RRB- and the beetle Tribolium -LRB- Ober and Jockusch 2006 -RRB- . While RNAi analysis of wg and hh in the cricket Gryllus -LRB- Miyawaki et al. 2004 -RRB- failed to reveal significant effects on segmentation , RNAi analysis of armadillo , a wg pathway component , does implicate wg signaling in segmentation in this short germ insect . In the long germ embryo of Drosophila , where all segments initiate virtually simultaneously , loss of any one of these three genes destabilizes the expression of the others and they eventually fade , resulting in shorter embryos that , in addition to the loss of segmental grooves , also show misspecified epidermal cell fates -LRB- Ingham et al. 1991 ; Forbes et al. 1993 -RRB- . We have investigated genes that encode the Hh-signaling pathway components hedgehog -LRB- hh -RRB- , patched -LRB- ptc -RRB- , smoothened -LRB- smo -RRB- , and cubitus interruptus -LRB- ci -RRB- to determine whether they function with en and wg in the formation of segmental boundaries in this short germ band insect . The Hh-signaling pathway is well-conserved between insects and vertebrates -LRB- Huangfu and Anderson 2006 -RRB- and is thus likely to be conserved in Tribolium . The main components were first elucidated in Drosophila , where Hh is secreted by cells in the posterior compartment of embryonic segments and larval imaginal discs . It diffuses to the anterior compartment -LRB- Lee et al. 1992 ; Tabata et al. 1992 ; Tashiro et al. 1993 -RRB- where the signal is controlled by two membrane proteins : Patched -LRB- Ptc -RRB- , a twelve pass transmembrane protein -LRB- Hooper and Scott 1989 ; Nakano et al. 1989 -RRB- and Smoothened -LRB- Smo -RRB- , a seven pass transmembrane protein -LRB- Alcedo et al. 1996 ; van den Heuvel and Ingham 1996 -RRB- . In the absence of Hh signal , Ptc represses Smo activity -LRB- Chen and Struhl 1996 -RRB- . Signaling is initiated by binding of Hh to its receptor Ptc , which relieves this repression and allows Smo to signal to a multimeric complex inside the cell . This complex is composed of the serine threonine kinase Fused -LRB- Alves et al. 1998 -RRB- , the kinesin related protein Costal-2 -LRB- Sisson et al. 1997 -RRB- , a novel cytoplasmic protein Suppressor of fused -LRB- Monnier et al. 1998 -RRB- and a zinc finger transcription factor Cubitus Interruptus -LRB- Ci ; Motzny and Holmgren 1995 -RRB- . In unstimulated cells , this complex sequesters ci , inhibits nuclear import of the full-length 155 kDa protein and promotes its cleavage to generate an N-terminal 75-kDa form containing the Zn finger DNA-binding domain , which can enter the nucleus and repress transcription of Hh target genes -LRB- Aza-Blanc et al. 1997 -RRB- . When Hh signal is transduced , activation of Smo inhibits ci cleavage and activates the full-length protein , which then translocates to the nucleus , resulting in the transcription of Hh-responsive target genes including wg , ptc , gooseberry , and decapentaplegic -LRB- Alexandre et al. 1996 ; Dominguez et al. 1996 ; Hepker et al. 1997 ; Ingham and McMahon 2001 -RRB- . Consistent with reports on other arthropods , we found that the expression patterns of hh , ci , smo , and ptc were largely conserved in Tribolium . Using RNAi to study the function of these genes during segmentation in Tribolium , we followed embryonic development in these embryos using En as a marker of segment development and integrity . When the Hh signal was depleted by RNAi , segments were specified normally in the posterior growth zone and the embryos elongated as fully as wild-type . En and wg expression in these embryos , although properly initiated , was not maintained and defects appeared during germ band retraction , resulting in tiny , sphere-shaped embryos lacking segmental grooves . On the other hand , overactivation of the pathway by ptc RNAi produced embryos with transient ectopic segmental grooves and embryonic cuticles with enlarged heads and thoracic appendages . All together , these results indicate that Hedgehog signaling is an essential component of the segment polarity network in Tribolium , which is necessary to maintain segmental integrity during germband retraction after the segments have been enumerated in the growth zone . The conserved function of an en -- wg -- hh gene circuit during segmentation suggests that the segment polarity genes constitute a robust gene regulatory module in this short germ insect . Materials and methods Beetle husbandry Tribolium castaneum strain GA-1 was reared in whole wheat flour supplemented with 5 % dried yeast at 30 C -LRB- Beeman et al. 1989 -RRB- . Identification of hedgehog pathway component genes in the Tribolium genome Partial cDNAs of tc-hh , tc-ci , tc-smo and tc-ptc , cloned into the pCR4TOPO vector -LRB- Invitrogen -RRB- , were obtained from Y. Tomoyasu . Orthologs of each gene were identified in the annotation of the Tribolium genome -LRB- the Tribolium genome consortium , in review -RRB- . The sequences of the partial cDNAs matched those deduced from the gene models with minor differences . hh , glean gene number tc01364 or NCBI mRNA accession number XM961615 , is located on LG 2 ; smo , TC05545 or XM966834 , is on LG 8 ; ci , TC03000 or XM965017 , is on LG 3 and ptc , TC04745 or XM962700 is on LG 1 = X . In situ hybridization and immunostaining Whole mount in situ hybridizations were performed according to established protocols -LRB- Tautz and Pfeifle 1989 -RRB- . Expression of Engrailed in Tribolium embryos was determined using the α-Invected antibody , 4D9 which cross-reacts with Tc-En -LRB- Brown et al. 1994 -RRB- . Double staining for the different mRNAs in addition to En protein was performed simultaneously according to the protocol of Nagaso et al. -LRB- 2001 -RRB- . RNA interference -LRB- RNAi -RRB- Templates for dsRNA synthesis were amplified as described -LRB- Tomoyasu et al. 2007 -RRB- . Double stranded RNA was synthesized using the T7 MEGAscript kit -LRB- Ambion -RRB- and purified using the MEGAclear kit -LRB- Ambion -RRB- . Different amounts of dsRNA -LRB- Table 1 -RRB- were mixed with injection buffer -LRB- 5 mM KCl , 0.1 mM KPO4 pH 6.8 -RRB- prior to injection . Parental RNAi was performed and affected embryos were analyzed as previously described -LRB- Bucher et al. 2002 -RRB- . Microscopy and imaging Stained embryos and larval cuticles were documented with a Nikon Digital DXM 1200F camera on an Olympus BX50 microscope using Nikon ACT-1 version 2.62 software . Brightness and contrast of all images were adjusted and some were placed on a white background using Adobe Photoshop 7.0.1 software . Table 1 Summary of RNAi effects Gene DsRNA μg/μl Class I -LRB- % -RRB- Class II -LRB- % -RRB- Class III -LRB- % -RRB- Undeveloped -LRB- % -RRB- Wild type -LRB- % -RRB- Totals -LRB- n -RRB- Tchh 3.0 0 6 83 11 0 464 1.5 18 73 0 9 0 477 Tcsmo 3.0 0 0 89 11 0 579 1.5 0 43 43 14 0 477 0.75 38 44 0 20 0 230 Tcci 3.0 0 33 61 5 0 694 1.5 0 30 63 7 0 405 0.75 73 19 0 8 0 293 Tcptc 4.0 0 76 -- 24 0 271 2.0 13 68 -- 19 0 189 Embryos were collected every 48 h for the first 2 weeks after injection . Phenotypic classes are unique for each gene . See text for details . Percentages have been rounded up . Results Expression patterns of Tc-hh , Tc-smo , and Tc-ci Tc-hh transcripts are first detected in the presumptive head lobes on either side of the ventral mesoderm -LRB- arrowhead in Fig. 1a -RRB- and at the posterior end of the embryo . As the embryonic rudiment condenses , faint stripes of Tc-hh expression appear immediately posterior to the intense stripes in the head lobes and in the presumptive mandibular segment -LRB- arrowheads in Fig. 1b -RRB- . Gnathal and trunk stripes appear in an anterior to posterior progression -LRB- Fig. 1c -RRB- . Double staining for En expression revealed that Tc-hh and Tc-En are coexpressed in cells of the posterior compartment in each segment -LRB- Fig. 1d -RRB- . During germband elongation , expression at the posterior end of the embryo resolves into spots on either side of the mesoderm and eventually into a ring surrounding the proctodeum -LRB- Fig. 1b -- d and arrowhead in f -RRB- . In the head , twin spots appear on either side of the presumptive stomodeum . As the head lobes mature , expression in the anterior region of the developing stomodeum increases -LRB- Fig. 1c , d and black arrowhead in e -RRB- , while the anterior-most stripes of Tc-hh expression resolve into spots in the brain that overlap with the Tc-En-expressing cells -LRB- Fig. 1c , d and blue arrowhead in e -RRB- . Expression at the posterior end of the embryo continues throughout germ band extension -LRB- Fig. 1b , d and f -RRB- eventually surrounding the proctodeum as it invaginates -LRB- Fig. 1f -RRB- . As segments mature , Tc-hh expression at the ventral midline clears and Tc-hh is not expressed in the CNS . In total , there are three gnathal , three thoracic , and ten abdominal Tc-hh stripes with additional expression in the stomodeum , proctodeum , antennae , and brain -LRB- Fig. 1d -RRB- . Fig. 1Expression of Tc-hh , Tc-ci , and Tc-smo in Tribolium during segmentation . In these ventral views , anterior is to the left . a -- cTc-hh ; d -- fTc-hh -LRB- purple -RRB- and Tc-En -LRB- gold -RRB- ; g -- hTc-smo ; i -- kTc-ci ; l , mTc-ci -LRB- purple -RRB- and Tc-En -LRB- gold -RRB- ; a Anterior stripes of Tc-hh -LRB- arrowhead -RRB- in the presumptive head lobes of a blastoderm embryo . Expression at the posterior end of the embryo is not in the plane of focus . b Weak stripes appear in the antennal and mandibular segments -LRB- arrowheads -RRB- posterior to the dark stripes in the head lobes . Twin spots of expression flank the mesoderm at the posterior end of the embryo and near the stomodeum . c In addition to expression in the head lobes and antennae , three gnathal stripes and one trunk stripes appear in an elongating germband embryo . d Coexpression of Tc-En -LRB- gold -RRB- and Tc-hh -LRB- purple -RRB- in the posterior compartment of each segment . Note Tc-En expression at the ventral midline in the absence of Tc-hh . e -- fTc-hh expression in the stomodeum and the proctodeum -LRB- arrowheads -RRB- in the absence of Tc-En . Expression of Tc-hh in the head lobes has resolved into spots that overlap Tc-En expression -LRB- blue arrowhead -RRB- . g Ubiquitous expression of Tc-smo in an early germ band embryo . h In an elongating germ band embryo , Tc-smo expression appears ubiquitous with some segmental modulation -LRB- arrowheads -RRB- . i Expression of Tc-ci at the anterior edge of the head lobes -LRB- arrowhead -RRB- of a young germband embryo . j Gnathal stripes appear first in a slightly older embryo . Expression throughout the posterior region of the embryo fades anteriorly . k Wide Tc-ci stripes in the head lobes and antennal segments as well as in the gnathal and thoracic segments of an elongating germ band embryo . l Tc-En -LRB- gold -RRB- expression does not overlap Tc-ci -LRB- purple -RRB- expression in cells of the anterior compartment of each segment . m Enlarged view of a few thoracic segments from the embryo shown in l reveals a gap of two or three rows of cells anterior to the Tc-En stripes that do not express Tc-ci -LRB- arrowhead -RRB- Tc-smo is expressed ubiquitously in the germband -LRB- Fig. 1g -RRB- in Tribolium . As the germ band elongates , expression appears to modulate slightly within each segment -LRB- arrowheads in Fig. 1h -RRB- . smo transcripts are expressed in a similar manner in Drosophila -LRB- Alcedo et al. 1996 -RRB- . Tc-ci expression is first detected at the anterior edge of each head lobe in the early embryonic rudiment -LRB- arrowhead in Fig. 1i -RRB- . Dynamic expression of Tc-ci in the head lobes eventually resolves to a wedge of cells in the lateral eye field -LRB- Fig. 1j -RRB- . Expression is detected in the labrum -LRB- Fig. 1l -RRB- and in a broad posterior region , which fades toward the anterior -LRB- Fig. 1j -RRB- . As the germ band elongates , Tc-ci is expressed in a broad stripe in every segment in an anterior to posterior progression -LRB- Fig. 1i -- k -RRB- . Double staining for Tc-En indicates that Tc-ci is expressed in the anterior compartment where the anterior-most Tc-ci expressing cells are immediately posterior to the Tc-En-expressing cells of the preceding segment -LRB- Fig. 1m -RRB- . Segmentally reiterated stripes of ci expression are also observed in the mulberry silkworm Bombyx mori -LRB- Dhawan and Gopinathan 2002 -RRB- , the spider Cupiennius salei -LRB- Damen 2002 -RRB- , and the millipede Glomeris -LRB- Janssen et al. 2004 -RRB- , suggesting that the segmental expression of ci in the anterior region of each segment is conserved among arthropods . Closer examination of Tc-ci expression revealed a gap of two or three rows of cells immediately anterior of the Tc-En-expressing cells that do not express Tc-ci -LRB- Fig . lm -RRB- . In contrast , expression of ci throughout the anterior compartment of each segment in Drosophila is thought to be essential to the function of the Hh-signaling pathway in maintaining wg expression -LRB- Orenic et al. 1990 ; Hepker et al. 1997 -RRB- . Expression of ci immediately anterior to En-expressing cells is conserved in the spider -LRB- Damen 2002 -RRB- , but has not been reported for Bombyx or Glomeris . Thus , it is not clear whether this unusual expression pattern is unique to Tribolium . RNAi analysis of Tc-hh , Tc-smo , and Tc-ci The conserved expression patterns of Tc-hh and Tc-smo are consistent with the hypothesis that the Hh function in segmentation is conserved in Tribolium , but the lack of Tc-ci gene expression in cells immediately anterior to Tc-hh is not . To determine whether the Hh-signaling pathway is required for proper segmentation , we performed parental RNAi analysis of these Hh pathway components . Three different amounts of Tc-hh and Tc-smo dsRNA were injected , which uncovered a range of hypomorphic phenotypes -LRB- Table 1 -RRB- . Similar RNAi phenotypes were produced for both genes , and the RNAi cuticles were classified into three different categories -LRB- Class I , II , and III -RRB- based on severity as listed in Table 1 . Regardless of the severity , none of the RNAi embryos hatched and the cuticle had to be dissected out of the vitelline membranes . Class I cuticles display the weakest phenotypes , in which the head is severely reduced and contains only rudimentary limb structures . The legs are present but slightly warped , and segmental grooves in the abdomen are occasionally fused but all eight abdominal segments are present -LRB- Fig. 2b -LRB- Tc-hh -RRB- , f -LRB- Tc-smo -RRB- -RRB- . Class II cuticles are small and spherical , with a large protuberance at the anterior end and three small warped pairs of limbs . The presence of the spiracle on the second thoracic segment -LRB- arrowhead in Fig. 2c and g -RRB- allowed us to identify these structures as rudimentary legs . The small amount of cuticle posterior to these small warped legs is smooth and lacked any abdominal features -LRB- Fig. 2c -LRB- Tc-hh -RRB- , g -LRB- Tc-smo -RRB- -RRB- . The most severely affected embryos , Class III , produced small spherical cuticles with a large protuberance at the anterior end and no obvious head structures or thoracic limbs -LRB- Fig. 2d -LRB- Tc-hh -RRB- , h -LRB- Tc-smo -RRB- -RRB- . These cuticles are very smooth on all sides , with no sign of grooves and are quite small relative to the size of similarly aged wild-type cuticles -LRB- Fig. 2a Tc-hh , e Tc-smo -RRB- . The head and gnathal appendages appear to be more sensitive to the depletion of Tc-hh or Tc-smo than the legs , as even in the weakly affected individuals the head , including gnathal segments , failed to form properly -LRB- Fig. 2b , f -RRB- . Fig. 2Tc-hh , Tc-smo , and Tc-ci RNAi phenotypes in first instar larval cuticles . a -- dTc-hh RNAi ; e -- hTc-smo RNAi ; i -- lTc-ci RNAi . a Comparison of a severely affected small spherical Tc-hh RNAi cuticle and a wild-type cuticle at the same magnification . b Lateral view of a mildly affected Class I cuticle containing reduced gnathal appendages , normal legs and all abdominal segments . c Ventral view of a Class II cuticle that is similar in size and shape to more severely affected Class III cuticles but retains three pairs of thoracic appendages . Note spiracle on second thoracic segment -LRB- arrowhead -RRB- . d Ventral view of a small spherical Class III cuticle with rudimentary head structures , no appendages and no segmental grooves . e Comparison of a severely affected small spherical Tc-smo RNAi cuticle and a wild-type cuticle at the same magnification . f Class I cuticle with highly reduced head structures and mouthparts but a full complement of body segments . g Class II cuticle with three pairs of legs and rudimentary head structures . Note spiracle on second thoracic segment -LRB- arrowhead -RRB- . h A severely affected Class III Tc-smo RNAi cuticle phenotypically similar to the most severely affected Tc-hh RNAi cuticle . i Comparison of a severely affected small spherical Tc-ci RNAi cuticle and a wild-type cuticle at the same magnification . j Mildly affected Class I cuticle with reduced head structures , three pairs of legs and mildly fused abdominal segments . k Class II cuticles are slightly longer than intermediate Tc-hh or Tc-smo RNAi cuticles with highly fused abdominal segments . l A severely affected Class III Tc-ci RNAi spherical cuticle with three pairs of legs The phenotypes of Tc-ci RNAi embryos are not as severe as those observed for Tc-hh or Tc-smo . The most weakly affected , Class I Tc-ci RNAi cuticles -LRB- Fig. 2j -RRB- , are shorter and fatter than wild-type with severely reduced heads , but contain all thoracic and abdominal segments . The Class II Tc-ci RNAi cuticles are also shorter than wild-type with a protuberance at the anterior end and fairly normal thoracic limbs , but little to no sign of segmental grooves in the abdomen -LRB- Fig. 2k -RRB- . Similar to Class III RNAi embryos of Tc-hh and Tc-smo , Class III Tc-ci RNAi embryos produced smooth unsegmented cuticles lacking heads and gnathal appendages -LRB- Fig. 2l -RRB- that are considerably smaller than wild-type cuticles -LRB- Fig. 2i -RRB- . Unlike Tc-hh and Tc-smo Class III RNAi embryos , they still produced fairly normal legs -LRB- Fig. 2l -RRB- . Interestingly , loss of function mutants of ci in Drosophila also produce milder limb phenotypes than do hh mutants -LRB- Methot and Basler 1999 -RRB- . To understand how the segmentation process is affected by loss of Hh signaling , we followed Tc-En expression during elongation and retraction in Class III RNAi embryos -LRB- Fig. 3 -RRB- . In RNAi embryos for all three genes , segmental stripes of Tc-En initiate normally during germ band elongation -LRB- blue arrowheads in Fig. 3a , d and g -RRB- . Tc-En expression fades as the segments matured -LRB- black arrowheads Fig. 3a , d and g -RRB- , suggesting that Hh signaling is required for the maintenance of Tc-En . However , Tc-En expression in cells along the midline , presumably in the developing CNS -LRB- wild-type , Fig. 3i -RRB- , is maintained in the RNAi embryos , which allowed us to follow segmentation in RNAi embryos . Tc-hh , Tc-smo , and Tc-ci RNAi embryos completed elongation more or less normally -LRB- Fig. 3a , d and g -RRB- , but abnormalities became evident during retraction . The head lobes fail to mature , and there is no evidence of antennal Tc-En stripes . There is also a protuberance at the anterior end of the embryo that is likely to correspond to the anterior protuberance seen in the RNAi cuticles -LRB- Fig. 2d , h and l , and arrow in 3 h -RRB- . In embryos that completed retraction , the unsegmented germbands are highly compacted , and Tc-En expression pattern is very irregular -LRB- Fig. 3c , e and h -RRB- . Compared to wild-type germ bands at a similar stage , these germ bands occupy only a portion of the egg -LRB- Fig. 3j , k -RRB- . In addition , loss of early Hh signaling at the ventral midline affected cell fate , as indicated by the loss of Tc-En expression here -LRB- Fig. 3b , e -RRB- . Loss of Hh signal in Drosophila similarly affects En expression in midline cells -LRB- Bossing and Brand 2006 -RRB- . We also found that Tc-wg expression was initiated but not maintained in these embryos -LRB- data not shown -RRB- , suggesting Tc-wg is a target of the Hh pathway . Fig. 3En staining in wild-type and severely affected Tc-hh , Tc-smo and Tc-ci RNAi embryos during elongation and retraction . a -- cTc-hh RNAi ; d -- f , kTc-smo RNAi ; g , hTc-ci RNAi ; i , j wild-type . a Tc-En expression initiated normally in the posterior segments of an elongating Tc-hh RNAi germband embryo -LRB- blue arrowhead -RRB- but did not persist in older , more anterior segments -LRB- black arrowhead -RRB- . b During germband retraction , patches of Tc-En expression are visible near the ventral midline , and laterally in the posterior segments . c In a highly compacted germ band with fused segments , Tc-En expression is disrupted along the ventral midline . d Expression of Tc-En in an elongating Tc-smo RNAi embryo initiated normally -LRB- blue arrowhead -RRB- but failed to persist in older , more anterior segments -LRB- black arrowhead -RRB- . e During germband retraction , Tc-En is expressed in patches along the ventral midline , but has faded laterally . f In a highly compacted germ band , persists in a disrupted pattern along the ventral midline . g In an elongating Tc-ci RNAi embryo , similar to Tc-hh and Tc-smo RNAi , Tc-En initiated normally -LRB- blue arrowhead -RRB- but is not maintained -LRB- black arrowhead -RRB- . h Tc-En expression at the ventral midline of a retracted germband embryo . Note the extended stomodeum -LRB- arrowhead -RRB- near the anterior end . i Tc-En expression in the CNS along the ventral midline and in the lateral ectoderm of each segment of a wild-type embryo during germband retraction . j Lateral view of a wild-type embryo inside the vitelline membrane at the end of germ band retraction . Tc-En expression persists laterally to the edge of the germband . k Ventral view of a severely affected Tc-smo RNAi embryo located near one end of the egg hedgehog was first isolated in a screen for mutations that disrupt the Drosophila larval cuticle pattern and identified as one of the segment polarity genes with a ` lawn of denticles ' phenotype -LRB- Nusslein-Volhard and Wieschaus 1980 -RRB- . In segment polarity mutants -LRB- e.g. , wg , hh , and smo -RRB- , deletion of a portion of the larval epidermis in each segment is accompanied by a mirror image duplication of the remaining structures . As a result , they contain the normal number of segments , but are smaller than wild-type due to partial deletion of each segment . In our study , we found Tc-hh and Tc-smo RNAi embryos to be smaller in size than the wild-type Tribolium larva -LRB- Fig. 2a , e -RRB- , which may be due to cell death , fusion , or failure of cell division in segments during germ band retraction . The few random bristles produced in severely affected RNAi cuticles do not have any definable polarity suggesting that , unlike Drosophila , loss of function of these genes in Tribolium does not appear to produce mirror image duplications or affect polarity within the segments . Analysis of Tc-ptc and overactivation of the Hh pathway Transcripts of Tc-ptc are first detected in a broad domain in the posterior regions of the head lobes in the embryonic rudiment encompassing the antennal segments and the stomodeum , and at the posterior end of the embryo -LRB- Fig. 4a -RRB- . Broad segmental stripes of Tc-ptc appear in an anterior to posterior progression during germband elongation -LRB- Fig. 4b -RRB- . Expression fades in the middle of each initial stripe resulting in two narrow Tc-ptc stripes per segment -LRB- Fig. 4c -RRB- . Double staining with Tc-En and Tc-ptc indicates that in each segment , one of the narrow Tc-ptc stripes marks the anterior boundary of a segment while the other is located immediately anterior to Tc-En-expressing cells -LRB- Fig. 4c and arrowhead in inset -RRB- . This pattern persists even after germ band retraction ; the anterior stripe appears stronger than the posterior stripe in each segment . ptc expression during segmentation in Drosophila is similarly dynamic -LRB- Nakano et al. 1989 ; Hidalgo and Ingham 1990 -RRB- . Fig. 4Analysis of Tc-ptc in Tribolium.a -- cTc-ptc expression in wild-type embryos ; d -- fTc-ptc RNAi cuticles ; g wild-type ; h -- mTc-ptc RNAi . a Expression of Tc-ptc in three gnathal stripes , in a broad posterior region of the head lobes and in the posterior region of an early germ band embryo . b Segmental stripes appear sequentially during elongation and resolve in double stripes . c Tc-En -LRB- gold -RRB- expression in the posterior compartment abuts , but does not overlap the double Tc-ptc stripes -LRB- extent denoted by black lines -RRB- in the anterior compartment of each segment . Inset : enlarged view of Tc-ptc stripes adjacent to a stripe of Tc-En -LRB- arrowhead -RRB- . d A mildly affected Class I Tc-ptc RNAi cuticle with deformed legs and head appendages . e A more severely affected Class II cuticle with enlarged misshapen legs but a normal complement of abdominal segments . f Comparison of a Class II Tc-ptc RNAi cuticle and a wild-type cuticle at the same magnification . g Expression of Tc-En -LRB- gold -RRB- and Tc-wg -LRB- purple -RRB- in a wild-type embryo during elongation . h Segmental expression of Tc-En and Tc-wg initiated fairly normally in a Tc-ptc RNAi germband . Tc-wg expression domains in the head lobes and around the proctodeum are expanded . i Expanded expression of Tc-wg in a slightly older embryo . j Ectopic stripes of Tc-En expression -LRB- blue arrowhead -RRB- in a germband undergoing retraction . k Ectopic expression of Tc-En at the ventral midline -LRB- arrowhead -RRB- in a close up of the embryo in h. l Transient grooves -LRB- arrowheads -RRB- around expanded Tc-wg expression domains in a close up of the embryo in i. m Ectopic Tc-En stripes -LRB- blue arrowhead -RRB- between each set of normal Tc-En stripes -LRB- black arrowhead -RRB- in a close up of the embryo in j. T1 , first thoracic segment As discussed above , Ptc is a negative regulator of the Hh-signaling pathway in Drosophila . Depletion of a negative regulator of the pathway would ectopically activate the pathway . To understand what happens when the Hh pathway is overactivated in Tribolium , we performed parental RNAi using two different amounts of Tc-ptc dsRNA -LRB- Table 1 -RRB- . The resulting cuticles -LRB- Fig. 4d , e , f -RRB- are very different from the ones described above for the other three genes . In mildly affected embryos -LRB- Class I -RRB- , the head appendages are misshapen , while the legs are relatively normal -LRB- Fig. 4d -RRB- . In the most severely affected embryos -LRB- Class II -RRB- , all segments are present , but the head and thoracic appendages are enlarged and misshapen -LRB- Fig. 4e , f -RRB- . To understand the phenotype of the Tc-ptc RNAi embryos , we examined the expression of Tc-wg and Tc-En . In Tc-ptc-depleted embryos , segmental expression of Tc-En and Tc-wg is initiated normally -LRB- Fig. 4h -RRB- , although the domains of Tc-wg expression in the head and at the posterior end of the embryo are expanded . Closer inspection revealed ectopic Tc-En expression at the ventral midline -LRB- Fig. 4k -RRB- . In slightly older embryos , Tc-wg is expressed more intensely than normal in enlarged domains -LRB- Fig. 4i , l -RRB- . Cells surrounding the expanded Tc-wg domains appear to be invaginating , as if beginning to form grooves . Closer inspection revealed a row of non-Tc-wg-expressing cells between the Tc-wg domain and the grooves -LRB- Fig. 4l -RRB- . Tc-En is expressed in ectopic stripes that , in combination with the normal Tc-En stripes , would surround the expanded Tc-wg domains -LRB- Fig. 4j , m -RRB- . In addition to the normal grooves that form posterior to the normal Tc-En stripe , ectopic grooves initiate anterior to the ectopic Tc-En stripes . Thus , overactivation of the Hh-signaling pathway in Tribolium leads to overexpression of Tc-wg , and ectopic expression of Tc-En , which results in ectopic groove formation . In Drosophila , binding of Hh to Ptc receptor relieves repression of wg and allows expression of target genes . In Drosophila ptc mutants , ectopic induction of wg and En result in the formation of extra grooves -LRB- Nakano et al. 1989 -RRB- , suggesting that the functional role of Ptc is highly conserved between these two species . However , unlike Drosophila , the ectopic grooves in Tribolium are transient and it appears that a late regulatory action restores the normal number of segments in this insect . Discussion Expression patterns suggest that the function of the Hh-signaling pathway is conserved in several developmental pathways in Tribolium . In Tribolium , the expression patterns of the four Hh-signaling pathway components we examined are highly similar to those of their Drosophila counterparts . In both Drosophila and Tribolium , the domains of Tc-hh are very similar to those of Tc-En . However , there are some significant differences in their expression dynamics . For example , the antennal Tc-En stripes appear after the three gnathal Tc-En stripes , whereas antennal Tc-hh stripes appear before the gnathal Tc-hh stripes . Each metameric stripe of Tc-hh is laterally continuous across the width of the germband after mesoderm invagination . Later , Tc-hh expression has disappeared at the ventral midline in anterior segments , while in posterior segments the stripes are still continuous . In contrast , Tc-En expression continues in the CNS after Tc-hh expression has faded there . Tc-hh expression in the stomodeum and at the posterior end of the blastoderm embryo in the absence of Tc-En suggests that , similar to hh in Drosophila -LRB- Lee et al. 1992 ; Mohler and Vani 1992 ; Tashiro et al. 1993 -RRB- , Tc-hh is involved in some En-independent processes in these regions . In both Drosophila and Tribolium , expression of segment polarity genes is initiated by pair-rule genes -LRB- recently reviewed by Damen 2007 -RRB- . Soon thereafter , their expression is controlled by interactions between the segment polarity genes themselves . In Drosophila , Ptc is constitutively active unless or until Hh represses Ptc activity . In the absence of Hh , unbound Ptc keeps the pathway switched off . One of the targets of the Hh pathway is ptc itself . In cells where its activity is antagonized upon binding of Hh , ptc continues to be expressed , but ptc expression disappears in cells that do not receive the signal . Thus , an initially broad domain of ptc expression resolves into two narrow stripes flanking the En expression domain . Tc-ptc expression pattern is similar : each broad stripe splits into two , such that each En stripe is bracketed by two Tc-ptc stripes , suggesting that Tc-ptc itself might also be a target of the Hh pathway in Tribolium . The expression patterns of Hh pathway component genes are highly conserved between Drosophila and Tribolium , except for that of Tc-ci -LRB- Fig. 1l , m -RRB- . In Drosophila , ci transcripts are initially expressed uniformly in the early cellular blastoderm and persist until the end of germ band elongation . At that point , ci expression is directly repressed by En in cells of the posterior compartment in each segment . In Tribolium , expression of Tc-ci is somewhat different in that there is a narrow region of two to three cells between the Tc-ci and En-expressing cells that does not express Tc-ci . In Tribolium , the absence of Tc-ci transcripts in cells just anterior to En-expressing cells might suggest the existence of an En-independent mechanism regulating Tc-ci expression . Alternatively , it is possible that Tc-ci transcripts turn over rapidly in these cells , but the expression pattern is conserved at the protein level . In Drosophila , ci is regulated post-transcriptionally . At stage 11 , ci transcripts are localized throughout the anterior compartment of each segment whereas protein levels are lower at the center of each transcriptional stripe and higher in cells that bracket the En-expressing cells -LRB- Motzny and Holmgren 1995 ; Slusarski et al. 1995 -RRB- much like what we describe here for ptc transcription in Tribolium and previously for Ptc protein levels in Drosophila . It will be interesting to see if Tc-ci is also post-transcriptionally regulated in Tribolium . Functional analysis of Hh pathway component genes supports conserved roles in segment boundary formation . In Drosophila , mutations in genes encoding positive regulators of the Hh pathway including hh , smo , and ci produce smaller than wild-type embryos with asegmental phenotypes -LRB- Nusslein-Volhard and Wieschaus 1980 -RRB- . In Tribolium , Tc-hh , and Tc-smo RNAi embryonic phenotypes are nearly identical ; both produce highly compacted spherical cuticles with no evidence of appendages or segmental grooves . The most severe Tc-ci RNAi phenotypes are not as severe as those of Tc-hh or Tc-smo RNAi . Interestingly , the ci94 allele in Drosophila is a null allele -LRB- Slusarski et al. 1995 ; Methot and Basler 1999 -RRB- and these mutant embryos differ considerably from hh mutants . hh mutants are much shorter in length and have a continuous ` lawn of denticles ' phenotype whereas ci94 mutants are almost normal in size and have alternating naked cuticle and denticle belts on the ventral surface . Target genes of hh are partially derepressed in the absence of ci , producing a ci phenotype that is milder than that of hh -LRB- Methot and Basler 2001 -RRB- . An analogous situation has been described for the Wingless signaling pathway in Drosophila where derepression of target genes in the absence of pangolin results in a milder segment polarity phenotype compared to that of wg null mutants -LRB- Cavallo et al. 1998 ; Waltzer and Bienz 1998 -RRB- . In hh and smo mutants , only the repressor function of ci remains , producing the catastrophic phenotype . In contrast , loss of both the activating and repressor forms results in the milder phenotype seen in ci null mutants . In Tribolium , the most severe Tc-ci RNAi phenotype is milder than the most severe Tc-hh RNAi phenotype , suggesting similar regulation of the Hh pathway in the beetle . In Drosophila , loss of Wg or Hh-signaling results in larvae that are smaller than wild-type due , at least in part , to epidermal cell death during and after germband retraction -LRB- Martinez-Arias and Lawrence 1985 -RRB- or a combination of transformation and cell death -LRB- Klingensmith et al. 1989 -RRB- . In Tribolium , hh , smo , and ci RNAi individuals are greatly reduced in size compared to the wild-type -LRB- Fig. 2 -RRB- . These embryos go through the events of early embryogenesis normally , producing the full complement of segments and initiating Tc-En and Tc-wg expression . Tc-En and Tc-wg expression fade and the segmental remnants become highly compacted along the anterior -- posterior axis during retraction . Tribolium embryos lacking Tc-wg also elongate normally but fail to maintain En expression and form shorter than wild-type embryos during retraction -LRB- Ober and Jockusch 2006 -RRB- . While it is likely that cell proliferation and programmed cell death both contribute to shaping the embryo in Tribolium , during normal development cells divide randomly throughout the elongating germ band -LRB- Brown et al. 1994 -RRB- ; organized patterns of cell division or cell death have not been reported . Similarly , it is likely that excessive cell death or the lack of cell proliferation contributes to the severely compacted terminal phenotype of Tc-hh , Tc-smo or Tc-ci RNAi embryos , but closer examination will be required to determine if this is so . In Drosophila ptc mutants , deletion of the midregion of each segment is accompanied by a mirror image duplication of the remaining denticles -LRB- Nusslein-Volhard and Wieschaus 1980 -RRB- . In contrast , Tribolium Tc-ptc RNAi embryos , which also display the correct number of segments , are characterized by distended gnathal and thoracic appendages . Although there are some random bristles , we could not identify any noteworthy difference in the polarity of these bristles that could be attributed to a characteristic loss of function phenotype for genes belonging to this class . This suggests that unlike Drosophila , the loss of segment polarity gene function in Tribolium does not result in any morphologically identifiable polarity defect in the cuticle . In the absence of ptc in Drosophila -LRB- DiNardo et al. 1988 -RRB- and Tribolium , En expression is established properly ; but later , de novo En stripes appear between the normal stripes . Ectopic expression of En in Drosophila is not due to regulation of ptc by pair-rule factors -LRB- DiNardo et al. 1988 -RRB- . In the absence of ptc , the wg expression domain broadens anteriorly . Expanded wg expression induces ectopic En stripes , which cause ectopic groove formation , suggesting that the principal function of Ptc is to repress wg . Similar expression of Tc-ptc in Tribolium , considered with the expanded Tc-wg domains that are surrounded by ectopic Tc-En-expressing cells and the ectopic grooves transiently formed in Tc-ptc RNAi embryos suggest that the role of Tc-ptc is likely to be functionally conserved between Drosophila and Tribolium . However , in Tc-ptc RNAi embryos , ectopic Tc-En is also detected along the ventral midline , a phenotype that has not been described for Drosophila ptc mutants . hh RNAi has been attempted in the orthopteran Gryllus bimaculatus -LRB- Miyawaki et al. 2004 -RRB- . Unfortunately , this organism seems to be resistant to hh dsRNA . In the RNAi embryos , the level of hh is not reduced . The embryos develop normally and hatched larvae show no cuticular defects . Lack of ptc analysis in other insects makes it difficult to speculate as to whether this novel expression of Tc-En along the ventral midline is specific to Tribolium , or a general feature related to short germ development . Finally , although ectopic grooves appear to form around the expanded Tc-wg domains , they are not detected in the terminal cuticles , implying that events late in embryogenesis restore the normal number of segmental grooves . Conservation of the hh -- wg -- en gene circuit in short germ segmentation Several lines of evidence suggest that the segment polarity gene circuit , in which the expression of the hh , wg , and en genes are dependent upon one another in the long germ mode of segmentation elucidated in Drosophila , is conserved in the short germ mode of segmentation found in Tribolium . As in Drosophila , in the absence of smo , hh , ci -LRB- this paper -RRB- , or wg -LRB- Ober and Jockusch 2006 -RRB- in Tribolium , En expression is not maintained . In addition , Tc-wg mRNA fails to persist in Tc-hh , Tc-smo and Tc-ci RNAi embryos -LRB- data not shown -RRB- . Furthermore , expression patterns and RNAi phenotypes of the Hh pathway components we examined -LRB- Tc-hh , Tc-smo , Tc-ci , and Tc-ptc -RRB- suggest that the regulation of the Hh pathway is also conserved . Segment polarity genes , which function last in the segmentation gene hierarchy , are expressed in segmental fields that have been predefined by genes at higher levels -LRB- gap and pair rule -RRB- . Conservation of the segment polarity gene circuit in Tribolium suggests that that segment polarity genes form a robust regulatory module in the short germ mode of segmentation in this beetle and their expression patterns in numerous insects and chilicerates suggest that this module is likely to be conserved among the Insecta , and perhaps the Arthropoda . While the expression patterns of segment polarity orthologs are highly conserved , the expression patterns of pair-rule gene orthologs vary greatly among insects and other arthropods -LRB- recently reviewed in Tautz 2004 ; Peel et al. 2005 ; Damen 2007 -RRB- . Functional analysis in Tribolium indicates that interactions among pair-rule gene homologs differ from those of their Drosophila counterparts -LRB- Choe et al. 2006 -RRB- and that some secondary pair-rule genes function in opposite parasegmental registers in Drosophila and Tribolium -LRB- Choe and Brown 2006 -RRB- . These findings suggest that the inputs from pair-rule genes to the segment polarity gene module are likely to be quite different in each of these insects . Functional analysis of how pair-rule genes regulate the alternating expression of en and wg in Tribolium will provide insight into these differences , which will ultimately help us understand the evolution of genetic regulatory networks . Interestingly , the segment polarity gene module seems to be resilient enough to withstand such evolutionary changes in input from the pair-rule gene module .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hedgehog
smoothened
cubitus interruptus
patched
segmentation


Cisplatin-DNA adduct formation in patients treated with cisplatin-based chemoradiation : lack of correlation between normal tissues and primary tumor Purpose In this study , the formation of cisplatin-DNA adducts after concurrent cisplatin-radiation and the relationship between adduct-formation in primary tumor tissue and normal tissue were investigated . Introduction Concurrent chemoradiotherapy is more effective than radiotherapy -LRB- RT -RRB- alone , both in in vitro studies -LSB- 1 , 2 -RSB- as well as in clinical studies in many different tumor types , including advanced head and neck squamous cell carcinoma -LRB- HNSCC -RRB- and cervical cancer , leading to improvements in locoregional control and/or survival -LSB- 11 , 13 , 21 , 28 -RSB- . In a metaanalysis on concurrent chemoradiation in HNSCC , the addition of concurrent single agent cisplatin to RT was the most effective treatment regime with the largest improvement on overall survival -LSB- 5 -RSB- . Concurrent cisplatin-based chemoradiation is now considered standard care in advanced-stage HNSCC and cervical cancer . In addition to the increased efficacy of the combined treatment , it was shown that the concurrent regimens are accompanied by higher acute toxicity rates compared to radiation alone -LSB- 11 , 21 -RSB- , with more severe mucositis and gastrointestinal toxicity . Since a substantial number of patients treated with concurrent chemoradiation still fail to respond to this toxic treatment , there is a need for an accurate predictive assay , based on which patients that are likely to respond to the therapy can be selected . This strategy may also provide a tool to individualize and tailor treatment , based on evaluation of the predictive assay , early during therapy . One potential predictive marker is the formation of cisplatin-DNA adducts , which are formed when cisplatin reacts with the cellular DNA by binding to nucleotides . The majority of adducts are either intrastrand adducts with cisplatin bound between two guanosine -LRB- GG -RRB- nucleotides or adenosine -- guanosine -LRB- AG -RRB- nucleotides -LSB- 8 -RSB- . Cisplatin-DNA adducts can be measured in tumor and normal tissue . The level of adducts has been shown to correlate with cytotoxicity in vitro -LSB- 34 -RSB- , and with response to therapy in patients -LSB- 3 , 17 , 32 , 35 -RSB- . In most of these studies -LSB- 3 , 32 , 35 -RSB- , adduct measurements were performed in the normal tissue , with the assumption that normal tissue can be used as surrogate marker for tumor . In our institute , study protocols with cisplatin-DNA adduct measurements are ongoing in patients with HNSCC and cervical cancer , who are all treated with concurrent cisplatin-based chemoradiation . The objectives of the current study are -LRB- 1 -RRB- to investigate the two major forms of cisplatin-DNA adducts -LRB- GG and AG adducts -RRB- after different schedules of cisplatin given concurrently with radiation and -LRB- 2 -RRB- to explore relationships between adducts in primary tumor and normal tissue . We specifically wanted to investigate whether the level of adducts in tumors are reflected by those in normal tissues . In studies focused on the predictive value of cisplatin-DNA adduct levels , this would then justify the use of more easily obtained normal tissues as a surrogate for tumor samples . Patients and methods Concurrent chemoradiation protocols This study on adduct formation was approved by the medical ethical committee of the participating hospitals . The main eligibility criteria were : patients scheduled for cisplatin chemoradiation , no previous treatment with cisplatin , and informed consent . Eligible patients were informed about the nature of the protocol and after written informed consent they were entered in the study . Patients were recruited from one of the following regimens . In advanced-stage HNSCC patients , two different cisplatin-based concurrent chemoradiation protocols -LRB- RADPLAT -RRB- were used . The RADPLAT 100 schedule , which is the most commonly administered schedule in HNSCC -LSB- 11 -RSB- , consisted of cisplatin given intravenously -LRB- IV -RRB- at a dose of 100 mg/m2 , as a 30 min infusion , 1 -- 2 h before RT at days 1 , 22 , and 43 of treatment . This treatment was part of a randomized trial on IV vs. intra-arterial chemoradiation . In RADPLAT daily LD , low-dose -LRB- LD -RRB- cisplatin was given as a 1 -- 2 min IV infusion at a dose of 6 mg/m2 daily , for a total number of 20 doses , 1 -- 2 h prior to RT. . This treatment was shown to be an effective alternative in HNSCC -LSB- 6 , 19 -RSB- . Patients ineligible for or refusing the randomized trial on intra-arterial chemoradiation were treated with RADPLAT daily LD , since this treatment could be given on an outpatient basis . The RT target volumes for all schedules included the primary tumor and the bilateral neck at a dose of 46 Gy in 23 fractions . A boost was given to the macroscopic tumor extensions at the primary tumor site and lymph node metastases at a dose of 24 Gy in 12 fractions , resulting in a total dose of 70 Gy in 35 fractions . In patients with advanced-stage squamous cell cervical cancer , concurrent chemoradiation -LRB- CERVIX 40 -RRB- consisted of weekly administration of cisplatin IV as a 4-h infusion at a dose of 40 mg/m2 , followed by RT within 1 -- 2 h . The total number of doses was 5 -- 6 , depending on external beam RT schedule and the number of intracavitary brachytherapy applications . The total radiation dose was usually 46 Gy to the cervical tumor , uterus , and pelvic lymph nodes , with a boost to the cervix tumor and other involved regions , to a total dose of 60 -- 74 Gy , depending on treated volume and whether or not intracavitary brachytherapy was given . Cisplatin-DNA adducts Before and after chemotherapy , normal tissue samples -LSB- white blood cells -LRB- WBC -RRB- and buccal cells -RSB- were collected . In patients with an accessible primary tumor , a biopsy of the tumor was also taken . To avoid harvesting necrotic tissue , the biopsy was taken at the viable peripheral rim of the tumor . Samples were obtained at different times , due to logistic reasons : for the patients in the RADPLAT 100 study , this was done 23 h after the end of administration of the first cisplatin infusion -LRB- given on day 1 of treatment -RRB- . In the patients in the CERVIX 40 study , samples were taken 20 h after the end of administration of the first weekly cisplatin infusion -LRB- given on day 1 of treatment -RRB- . For the patients in the RADPLAT daily LD group , samples were taken 1 h after the 5th dose on day 5 of treatment . WBC were isolated from whole blood samples according to a previously published protocol -LSB- 24 -RSB- . Buccal cells were collected in phosphate-buffered saline by scraping the bilateral buccal mucosa using a cotton swab . In HNSCC patients , the buccal mucosa could be located within the RT treatment fields , depending on the tumor site . The harvested cells were centrifuged -LRB- 5 min at 4 °C , 1,000 rpm -RRB- and resuspended in a Tris -- EDTA buffer and stored at − 80 °C until analysis . Tumor biopsies were taken and immediately frozen at − 80 °C until analysis . Quantification of GG - and AG-intrastrand adducts was performed by a 32P-postlabeling technique as previously described -LSB- 29 -RSB- . Internal standardization was incorporated in the present analysis method , by adding 300 fmol of TT nucleotides to each sample . From previous work , the reproducibility of the assay is known by analysis of duplicate specimens within the same experiment -LRB- within-run reproducibility -RRB- and by analysis of duplicate specimens in separate experiments -LRB- between-run reproducibility -RRB- -LSB- 29 -RSB- . The reproducibility was described for WBC and tumor samples and was within 10 % for the within-run precision and between 2 -- 20 % for the between-run precision . It was also determined for buccal cells in the same way and similar reproducibility was obtained . The concentration of DNA present in the samples was measured spectrophotometrically at 260 nm with the Nanodrop ND-1000 -LRB- Nanodrop Technologies Inc , Wilmington , DE , USA -RRB- . The cisplatin-DNA adduct levels were expressed as fmol/μg DNA . The lower limit of quantification for the Pt-GG and Pt-AG adducts was 0.087 and 0.053 fmol/μg DNA , respectively . Statistical analysis Analysis of data was performed in SPSS software -LRB- version 11.5 , SPSS , Inc. -RRB- . For quantitative comparison of numerical data between groups , the Student 's t-test was applied . The Pearson correlation coefficient and Spearman 's rank correlation coefficient were calculated for analysis of correlations between different samples -LRB- WBC , buccal cells , and primary tumor -RRB- on an intra-patient level . Results Samples for cisplatin-DNA adduct determination were obtained from 63 patients : 27 from RADPLAT daily LD , 15 from CERVIX 40 , and 21 from RADPLAT 100 . WBC samples were taken from 61 patients , buccal cells from 25 , and tumor biopsies from 23 of these patients . The reasons for the missing data for the normal tissue samples were : no collection of samples due to logistics or -LRB- in minority of cases -RRB- not sufficient volume for analysis . The reason for missing primary tumor biopsy data were : tumor not accessible for direct outpatient-based biopsy -LRB- in HNSCC patients -RRB- or refusal -LRB- in cervix cancer patients -RRB- . In WBC , all but three of the 60 available baseline samples were below the LLQ for the GG adducts and all but two below the LLQ for the AG adducts . This was probably due to some background signal inherent in the postlabeling method , since all patients had not been treated before with platinum chemotherapy . The yield of DNA , obtained from the buccal cell samples was rather low , ranging from 1 -- 10 μg . Baseline samples of buccal cells were available from 16 of 25 patients , of whom posttreatment samples were also available . The baseline values of GG adducts in buccal cells ranged from 0.067 to 0.745 fmol/μg DNA -LRB- mean 0.282 , SD 0.19 -RRB- and baseline values of AG adducts in buccal the cells ranged from 0.087 to 1.538 fmol/μg DNA -LRB- mean 0.398 , SD 0.38 -RRB- . All but two of the baseline GG-adduct values were above the LLQ and all the baseline AG-adduct values were above the LLQ . This was probably due to the low DNA quantities obtained from the buccal cell samples . The difference in adduct levels from baseline to post-infusion values was significant for the GG adducts , but not for the AG adducts . This implies that for measuring low quantities of AG adducts in the low amounts of buccal cell DNA available , we reached the limits of quantification with this postlabeling method . In Table 1 , the results are presented for the GG - and AG-adduct levels in normal tissue and primary tumor for the three different cisplatin-chemoradiation regimes . Adduct levels in primary tumor were two to five times higher than those in WBC for all three treatment regimes for both GG - and AG-adduct formation -LRB- Student t-test , P < 0.001 for both adduct types -RRB- . For the comparison of the adduct levels from the three different treatment protocols , the data from the RADPLAT daily LD were omitted , since the daily administration schedule and sampling time -LRB- 1 h after the infusion -RRB- were different from the other two regimens . The adduct levels in the RADPLAT 100 schedule were statistically significantly higher than those after the CERVIX 40 schedule for both tumors -LRB- Student t-test , P = 0.01 -RRB- and normal tissues -LRB- Student t-test , P < 0.01 -RRB- . Table 1Cisplatin-DNA adducts -LRB- in fmol/μg DNA -RRB- in normal tissue and primary tumor after different schedules of cisplatin-based chemoradiationTreatment scheduleWBCBuccal cellsTumorGGAGGGAGGGAGRADPLAT daily LDN2626111166 Mean0 .340.050.840.210.660.10 SD0 .100.190.390.140.370.05 CERVIX 40N1414771010 Mean0 .440.060.870.221.940.26 SD0 .170.030.260.061.470.26 RADPLAT 100N21217777 Mean1 .0470.1241.5630.3403.8660.413 SD0 .3770.0490.4340.0901.1010.089 See text for explanation of treatment schedulesN number of patients , SD standard deviation , WBC white blood cells , GG GG-adducts , AG AG adducts A highly significant linear correlation -LRB- Pearson correlation , r = 0.93 , P < 0.001 , n = 61 -RRB- was observed between the level of GG and AG adducts in WBC -LRB- see Fig. 1a -RRB- , with a mean ratio of GG/AG adducts of 7.6 ± 2.1 SD . Similar linear relationships and ratios were found for GG and AG adducts in primary tumor -LRB- r = 0.89 , P < 0.001 , n = 23 , and ratio 8.5 ± 2.8 ; Fig. 1b -RRB- and buccal cells -LRB- r = 0.85 , P < 0.001 , n = 23 , and ratio 3.9 ± 1.3 ; Fig. 1c -RRB- . Fig. 1Correlation-plots of GG - and AG-adduct levels in white blood cells -LRB- WBC -RRB- -LRB- panel a -RRB- , primary tumor biopsy -LRB- panel b -RRB- , buccal cells -LRB- panel c -RRB- . In each panel , the three different treatment groups are depicted : RADPLAT daily LD 5 × 6 mg -LRB- red squares -RRB- , CERVIX 40 mg -LRB- green circles -RRB- , and RADPLAT 100 mg -LRB- blue triangles -RRB- A trend was observed between GG-adduct levels in WBC and buccal cells , although not significant -LRB- r = 0.38 , P = 0.07 , n = 24 -RRB- . No significant correlations were found between tumor and normal tissue : tumor vs. WBC -LRB- r = 0.35 , P = 0.13 , n = 21 -RRB- and tumor vs. buccal cells -LRB- r = − 0.003 , P = 0.99 , n = 9 -RRB- . See Fig. 2 for scatter plots . Similar results were found for the AG adducts : no significant correlations were found between adducts in tumor vs. WBC -LRB- r = 0.14 , P = 0.55 , n = 21 -RRB- , tumor vs. buccal cells -LRB- r = 0.25 , P = 0.58 , n = 7 -RRB- or WBC vs. buccal cells -LRB- r = 0.26 , P = 0.24 , n = 22 -RRB- . Fig. 2Correlation plots of GG adducts in white blood cells -LRB- WBC -RRB- vs. buccal cells -LRB- panel a -RRB- , and normal tissue vs. tumor -LRB- panels b and c -RRB- . In each panel , the three different treatment groups are depicted : RADPLAT daily LD 5 × 6 mg -LRB- red squares -RRB- , CERVIX 40 mg -LRB- green circles -RRB- , and RADPLAT 100 mg -LRB- blue triangles -RRB- Discussion There are two main conclusions from the 63 patients included in these analyses . First , intra-tumoral adduct levels were substantially higher than those in normal tissue -LRB- WBC -RRB- at all cisplatin-dose levels examined . Second , no positive correlations were evident between adducts in tumors and normal tissues . It should be noted that the various schedules , the cisplatin doses , and the duration of infusions differed , as well as the sampling times . However , all analyses on adducts were performed on paired samples , within the same patient . This eliminates variance of these factors since the normal tissue and tumor samples all were obtained at similar time points after the cisplatin infusion . In the RADPLAT daily LD , some accumulation from the previous four daily 6 mg cisplatin infusions would have occurred and affected the day 5 measurement after the 5th infusion . In the RADPLAT 100 and CERVIX 40 patients , no such accumulation would have occurred , since the sampling was done 20 -- 23 h after the first infusion of cisplatin . Relatively little information is available regarding the in vivo formation of intra-tumoral cisplatin-DNA adducts in clinical series -LSB- 17 , 23 -RSB- . Most studies focused on intra-tumoral platinum concentrations , both in HNSCC -LSB- 12 , 33 -RSB- and cervical cancer patients -LSB- 15 , 22 -RSB- . These studies are mostly characterized by relatively low numbers of patients , probably due to the invasive nature of the procedure . The data on correlations between adducts and platinum content are contradictory : In an experimental study -LSB- 37 -RSB- , no relationship could be established between the intra-tumoral adduct levels and platinum content , although in one clinical study , a significant correlation was found -LSB- 23 -RSB- . Adduct levels in primary tumors were consistently two - to fivefold higher than in WBC . This was true for both GG and AG adducts . This finding was previously described in anecdotal clinical cases -LSB- 9 , 30 -RSB- . Similar observations were made in platinum content studies in an experimental tumor model -LSB- 18 -RSB- and in HNSCC patients -LSB- 33 -RSB- . Adducts in tumor were also higher than in buccal cells in the CERVIX 40 and RADPLAT 100 group , but not in the RADPLAT daily LD group . We observed a linear relationship between the two major adduct forms -LRB- AG and GG -RRB- for both normal tissues and tumor -LRB- Fig. 1a -- c -RRB- , although GG-adduct formation was 5 -- 12 times increased relative to the AG adducts , as reported earlier -LSB- 8 , 36 -RSB- . The linear relationships between GG and AG adducts serve as a validation of the assay . Apparently within a sample , both types of adducts are present in an equal proportion , although the absolute amounts differ greatly . In previous studies , the type of adduct responsible for the cytotoxic effect of platinum compounds has been investigated . From two of these studies -LSB- 7 , 36 -RSB- it was concluded that the AG adduct was responsible for the platinum cytotoxicity . From our study , such a conclusion can not be made , since both types of adducts were present in equal proportions . Adduct formation in different tissue samples showed a lack of correlation between tumor and normal tissue . One might have expected that higher adduct levels in normal tissue would be accompanied by more adducts in tumor , although this was not the case . Similar results were found in an animal study -LSB- 25 -RSB- and in a clinical series of uterine cervix cancer patients -LSB- 22 -RSB- , both demonstrating lack of correlation between tumor platinum and serum platinum concentrations . If adduct formation were merely a matter of cisplatin exposure , then a positive correlation would be expected . The reasons for the higher levels of adducts in tumor vs. normal tissue and the lack of correlation between them may be explained by differences between tumor and normal tissue in one of the following factors : Tumors are heterogeneous in terms of blood supply and perfusion , resulting in differences in cisplatin uptake and diffusion , drug-pumps may diminish intra-cellular cisplatin concentrations by active transmembrane transport of cisplatin , prohibiting adducts to be formed , and tumor cells may have less effective capacities to repair damage from cytotoxic agents . Adducts are formed rapidly and in a dose-dependent fashion within 1 -- 2 h after cisplatin exposure , with a gradual decrease -LRB- repair -RRB- within the next 20 -- 24 h -LSB- 20 , 32 , 36 -RSB- . The persistence of cisplatin-DNA adducts may therefore be regarded as a measure of repair and possibly be used as predictive assay . We therefore chose to measure adducts 20 -- 23 h after the end of chemotherapy infusion . Ideally , more frequent measurements would have generated more information on the rate of adduct formation , repair , and total exposure to adducts -LRB- like an AUC analysis -RRB- -LSB- 24 -RSB- . However , obtaining repeated biopsies is not feasible in clinical practice . The results from the buccal cell samples need to be interpreted with caution , especially the AG-adduct levels , since uncertainties remain . With the low quantities of AG-adducts in the low amounts of buccal cell DNA we could extract , we reached the limits of quantification of the postlabeling method . The rationale for measuring cisplatin-DNA adducts is that it could be used as a predictive assay : higher levels of adducts would predict favorable treatment outcome . Many studies have been performed for this purpose , investigating adducts in normal tissue -LRB- WBC and buccal cells -RRB- -LSB- 3 , 4 , 10 , 14 , 26 , 31 , 32 , 35 -RSB- . These study designs are based on the assumption that normal tissue can be used as a surrogate marker for tumor tissue with respect to cisplatin-DNA adduct formation . In our present study , however , we did not find such a correlation . This might explain why the results on the role of adduct formation to predictive outcome are heterogeneous and contradictory , as illustrated below . Several studies on adduct formation in WBC showed that the level of adducts was positively correlated with response to chemotherapy in patients with advanced disease in a variety of tumor sites -LSB- 31 , 32 -RSB- , while others showed no correlation -LSB- 4 , 26 -RSB- . One study showed a positive correlation in one tumor site -LRB- ovarian cancer -RRB- , but not in the other -LRB- breast cancer -RRB- -LSB- 14 -RSB- , and in another study the level of adduct formation showed a negative association with survival for day-5 adducts , while there was no difference for day-1 adducts -LSB- 10 -RSB- . In studies on adduct levels in buccal cells , a positive correlation was found between adducts and either disease response -LSB- 3 -RSB- or better survival in non-small cell lung cancer -LRB- NSCLC -RRB- -LSB- 35 -RSB- . We recently showed that adduct formation in primary tumor appeared to be associated with better progression-free survival in HNSCC -LSB- 17 -RSB- . Differences were observed between the levels of intra-tumoral adducts for the three chemoradiation schedules , with lower adducts after lower dosages of cisplatin . Based on this , however , one can not predict that the schedules with less adducts will result in less cytotoxicity , since not only the cisplatin dose , but also timing and schedule of cisplatin administration are crucial determinants of efficacy -LSB- 1 -RSB- . These factors may contribute to differences in the formation and rate of repair of adducts , resulting in different exposures to cisplatin-DNA adducts . These differences make it difficult to extrapolate from the observed adduct values in tumor and normal tissues to a prediction of superiority of one schedule over another . In future studies , immunohistochemistry on repair proteins like ERCC1 -LSB- 27 -RSB- or gene expression profiling studies on platinum resistance -LSB- 16 -RSB- could be used to improve the prediction of cisplatin sensitivity and prediction of therapy response . In conclusion , we have demonstrated that in concurrent chemoradiotherapy schedules , cisplatin adduct levels in tumors were significantly higher than in normal tissues -LRB- WBC -RRB- . No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies . This lack of correlation may , to some extent , explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as predictive test in anticancer therapy . Conflict of interest : none declared .

O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O 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chemoradiation
cisplatin
cervical cancer