摘要 目的 探讨8-B r-cAMP对人视网膜母细胞瘤HXO-Rb44细胞抑癌基因表达的效应。方法 应 用RNA斑点印迹技术检测细胞p16、p21wafl、wp53、mp53和 Rb的mRNA，应用免疫组化斑点印迹技术检测细胞P16、P21wafl 、PRb、cdk2、cdk4和PCNA蛋白质表达的免疫反应性(IR)。结果 在斑点 印迹标本上，人HXO-Rb44细胞p16、p21wafl、wp53及Rb 的mRNA信号和P16-IR、P21wafl-IR及PRb-IR均是 实验组高于对照组，P＜0.05～0.01，而mp53mRNA信号、PCNA-I R、cdk2-IR和cdk4-IR均是实验组低于对照组，P＜0.05～0.01 。结论 8-Br-cAMP上调人HXO-Rb44细胞p16、p21wafl、wp 53、Rb抑癌基因的表达及P16、P21wafl、PRb蛋白的表达， 并下调mp53mRNA、PCNA-IR、cdk2-IR和cdk4-IR的 表达，提示8-Br-cAMP可通过阻抑细胞周期进展相关基因的表达而抑制人HXO- Rb44细胞的生长增殖。
8-Br-cAMP up-reg ulates antioncogene expression in human retinoblastoma
HXO-Rb44 cells
Abstract ObjectiveIt is kno wn that 8-Br-cAMP is one of selective bi nding site analogues for cAMP RIIα to af fect cell growth through regulation of g ene expression．The p16，p21wafl，p53 a nd Rb are antioncogenes which affect cel l growth through control of cell cycle．T he aim of this study is to investigate t he 8-Br-cAMP effect on the expression of antioncogenes in human HXO-Rb44 cells． Methods Cultured HXO-Rb44 cells in RPMI -1 640 medium were divided into two aliquot s．8-Br-cAMP （2×10－5mol／L） was added i nto one aliquot for 24h as the experime ntal group（EG），the another aliquot witho ut 8-Br-cAMP as the control group（CG）．Af ter 24h，the cell suspension was dropped onto the nitrocellulose membrane．The mR NA of p16，p21wafl，wild type（w）p53，mut ant type（m） p53 and Rb were used respec tively with biotin-labeled cDNA probes b y intact cell RNA dot blot．The immunorea ctivity（IR） of P16，P21wafl，PRb，PCN A，cdk2 and cdk4 were detected respecti vely with specific monoclonal antibodies on dot blot．ResultsThe mRNA dot blot s ignals of mp53 and protein dot blot of cdk2-IR，cdk4-IR and PCNA-IR in EG were weaker than those in CG（P＜0.05～0.01）． W hile，the mRNA signals of p16，p21wafl，wp53 and Rb in EG were stronger than tho se in CG（P＜0.05～0.01）．The intensity of ea ch protein dot blot was consistent with that of their RNA dot blot （except for w P53-IR and mP53-IR not to be done）．Conc lusions（1）8-Br-cAMP could up-regul ate expression of antioncogenes includin g p16，p21wafl，wp53，Rb，and protein exp ression of P16，P21wafl and PRb．（2） 8-Br-cAMP could down-regulate mp53 gene expression and protein expression of cd k2，cdk4 and PCNA．The results suggest t hat 8-Br-cAMP could inhibit human HXO-Rb 44 cell growth through interfering rela ted gene expression of cell cycle．
Key words retinoblastoma antioncogenes dot blot cdks
Retinoblasto ma is one of common malignant tumors occ urred in children and ev en infants，it is also the study focus fo r international scientists．There were di fferent therapeutic methods，such as cryo therapy，photocoagulation，radiotherapy，s u rgery，chemotherapy，ect［1］．
8-Br-cAMP is one kind of analogues to combine with cAMP receptor RIIα subunit（8-Cl-cAMP a nd DB-cAMP also belong to the analogues） ．These analogues can inhibit the cell gr owth and facilitate cell differentiation through their selective binding sites t o regulate the gene expression［2］．The analogues have no toxicity，especially 8 -Br-cAMP，acting as gene expression regul ator to be used as one of method to trea t neoplasm．8-Br-cAMP has already been ap plied to Ⅱ stage of clinic therapy in ot her tumors including melanoma，breast can cer，adenocarcinoma，etc［3］．However，it has not been reported about the effects of 8-Br-cAMP on gene expression so far a ssociated retinoblastoma．This paper stud ied the effects of 8-Br-cAMP on expressi on of antioncogenes in human retinoblast oma HXO-Rb44 cells．
MATERIALS AND METHODS
Sample preparati on The cultured HXO-R b44 cells were divided into two aliquots ：(1)24h after addition of 8-Br-cAMP （Sig ma） in final concentration of 2×10－5m ol ／L to the medium，defined as the experime ntal group（EG）；(2)Without 8-Br-cAMP treat ment 24h a s the control group．Nitrocellulose membr ane ［NCM，Life Tech］ was used to blot the HXO-Rb44 cell suspensions in concentrat ion of 1×107 cells／ml．
Intact cel l RNA dot blot According to J Wu’s m ethod［4］ （Practical Protocols for Nonradiactive Biotech），the NCM with HXO-Rb44 cells for various RNA dot blots were treated for 2min with chloroform vapor to cleave t he cytomembrane of intact cells，subseque ntly denatured with RNA denaturing solut ion at 68℃ for 15min．After rinsing with 2×SSC，the samples were dried in an vacu um incubator at 80℃ for 2h．After hybrid ization respectively with bio-11-dUTP（Si gma） labeled cDNA probes，including p16 ，p21wafl，wp53，mp53 and Rb labeled cDNA probes by ‘Prime a Gene’kit （Promega），t he NCM as samples were washed gently wit h 0.1×SSC at 42℃ for 60min（4×15min）．Af ter blocking with acytelated BSA，the sam ples incubated with SA（Streptavidin）-AP（ alkaline phosphatase） conjugate （Promega ） were applied with AP color developing sy stem，NBT and BCIP used as the substrate． The negative controls included removal o f the labeled probe and predigestion of RNA with RNase（Promega） 100μg／ml at 37℃ for 60min．
Intact cell protein d ot blot After the NCM samples were b locked wit h 0.5％ Tween-20／TBS and treated with chl oroform vapor，the samples were incubated with the specific primary antisera to v arious genes expression products includi ng P16，P21wafl，Rb，PCNA，cdk2 and cdk4 resp ectively（Santa，DAKO or Zymed） and subseq uently incubated with AP conjugated seco ndary antibody．The color developing syst em was similar to intact cell RNA dot bl ot．
Signal detection All of t he signals on the NCM were scanned with 530 nm wav e length by a scanner （Shimadu）．The opti cal density（OD）was analysed by t test．
RESULTS
Our results showed that t he dot blot signals of p16，p21wafl，wp 53 and Rb antioncogenes in EG were all h igher than those in CG，P＜0.05～0.01，the signals of mp53 in EG was lower than t hat in CG（see Tab．1）．
Tab．1 The effect of 8-Br-cAMP on g ene expression of p16，p21wa fl，wp53， Rb
and mp53 in human H XO-Rb44 cells （ ±s ，n＝9）
| mRNA |
p16 |
p21 |
wafl |
wp53 |
Rbmp53 |
| EG |
2.69±0.91 |
2.43±0.57 |
3.47±1.12 |
2.89±1.01 |
1.44±0.87 |
| CG |
1.63±0.72 |
1.44±0.87 |
1.45±0.64 |
2.18±0.72 |
2.43±0.57 |
| P value |
＜0 .05 |
＜0.05 |
＜0.01 |
＜0.05 |
＜0.05 |
The immunost aining revealed that the dot blot immuno reactivity of P16，P21wafl and PRb protein expres-sion in human HXO-Rb44 ce lls were all stronger in EG than those i n CG，P＜0.05，but the PCNA-IR，cdk2-IR a nd cdk4-IR were weaker in EG than those in CG，P＜0.01．
Tab．2 The effect of 8-B r-cAMP on protein expression of P16，P 21wafl，PRb，PCNA，
cdk2 and cdk4 in h uman HXO-Rb44 cells （±s，n＝9）
| Protein |
P16 |
P21wafl |
PRb |
PCNA |
cdk2 |
cdk4 |
| EG |
2.27±0.60 |
2.86±0.75 |
2.84 ±0.59 |
1.28±0.48 |
2.51±1.52 |
1.81±1.00 |
| CG |
1.59 ±0.84 |
1.90±0.68 |
2.21±0.71 |
2.53±0.91 |
5.64±1.12 |
4.76±1.45 |
| P value |
＜0.05 |
＜0.05 |
＜0.05 |
＜0.01 |
＜0.01 |
＜0.01 |
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